Binding-triggered transcriptional switches and methods of use thereof

ABSTRACT

The present disclosure provides binding-triggered transcriptional switch polypeptides, nucleic acids comprising nucleotide sequences encoding the binding-triggered transcriptional switch polypeptides, and host cells genetically modified with the nucleic acids. The present disclosure also provides chimeric Notch receptor polypeptides, nucleic acids comprising nucleotide sequences encoding the chimeric Notch receptor polypeptides, and host cells transduced and/or genetically modified with the nucleic acids. The present disclosure provides transgenic organisms comprising a nucleic acid encoding a binding triggered transcriptional switch polypeptide and/or a chimeric Notch receptor polypeptide of the present disclosure. Binding triggered transcriptional switch polypeptides and chimeric Notch receptor polypeptides of the present disclosure are useful in a variety of applications, which are also provided.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/120,256, filed Feb. 24, 2015; U.S. Provisional PatentApplication No. 62/257,153, filed Nov. 18, 2015; and U.S. ProvisionalPatent Application No. 62/269,758, filed Dec. 18, 2015, whichapplications are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.EY016546; P50 GM081879; and R01 GM055040 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file,“UCSF-511WO_SeqList_ST25.txt” created on Jan. 26, 2016 and having a sizeof 649 KB. The contents of the text file are incorporated by referenceherein in their entirety.

INTRODUCTION

Notch receptors are transmembrane proteins that mediate cell-cellcontact signaling and play a central role in development and otheraspects of cell-to-cell communication, e.g. communication between twocontacting cells, in which one contacting cell is a “receiver” cell andthe other contacting cell is a “sender” cell. Notch receptors expressedin a receiver cell recognize their ligands (the delta family ofproteins), expressed on a sending cell. The engagement of notch anddelta on these contacting cells leads to two-step proteolysis of thenotch receptor that ultimately causes the release of the intracellularportion of the receptor from the membrane into the cytoplasm. Thisreleased domain alters receiver cell behavior by functioning as atranscriptional regulator. Notch receptors are involved in and arerequired for a variety of cellular functions during development and arecritical for the function of a vast number of cell-types across species.

SUMMARY

The present disclosure provides binding-triggered transcriptional switchpolypeptides, nucleic acids comprising nucleotide sequences encoding thebinding-triggered transcriptional switch polypeptides, and host cellsgenetically modified with the nucleic acids. The present disclosureprovides transgenic organisms comprising a nucleic acid encoding abinding-triggered transcriptional switch polypeptide of the presentdisclosure. Also provided are methods of locally modulating an activityof a cell using one or more binding-triggered transcriptional switchpolypeptides and a localized cell activation system using one or morebinding-triggered transcriptional switch polypeptides. Abinding-triggered transcriptional switch polypeptide of the presentdisclosure is useful in a variety of applications, which are alsoprovided.

The present disclosure provides chimeric Notch receptor polypeptides,nucleic acids comprising nucleotide sequences encoding the chimericNotch receptor polypeptides, and host cells genetically modified withthe nucleic acids. The present disclosure provides transgenic organismscomprising a nucleic acid encoding a chimeric Notch receptor polypeptideof the present disclosure. A chimeric Notch receptor polypeptide of thepresent disclosure is useful in a variety of applications, which arealso provided.

The present disclosure provides a chimeric polypeptide (also referred toherein as a “chimeric Notch receptor polypeptide”) comprising, fromN-terminal to C-terminal and in covalent linkage: a) an extracellulardomain comprising a first member of a specific binding pair; b) a Notchreceptor polypeptide, wherein the Notch receptor polypeptide has alength of from 50 amino acids to 1000 amino acids, and comprises one ormore ligand-inducible proteolytic cleavage sites; and c) anintracellular domain, wherein the first member of the specific bindingpair is heterologous to the Notch receptor polypeptide, and whereinbinding of the first member of the specific binding pair to a secondmember of the specific binding pair induces cleavage of the Notchreceptor polypeptide at the one or more ligand-inducible proteolyticcleavage sites, thereby releasing the intracellular domain. In somecases, the Notch receptor polypeptide has a length of from 300 aminoacids to 400 amino acids. In some cases, the chimeric Notch receptorpolypeptide comprises a linker interposed between the extracellulardomain and the Notch receptor polypeptide. In some cases, theintracellular domain is a transcriptional activator. In some cases, theintracellular domain is a transcriptional repressor. In some cases, theintracellular domain is a site-specific nuclease. In some cases, thesite-specific nuclease is a Cas9 polypeptide. In some cases, theintracellular domain is a recombinase. In some cases, the intracellulardomain is an inhibitory immunoreceptor. In some cases, the intracellulardomain is an activating immunoreceptor. In some cases, the first memberof the specific binding pair comprises an antibody-based recognitionscaffold. In some cases, the first member of the specific binding paircomprises an antibody. In some cases, where the first member of thespecific binding pair is an antibody, the antibody specifically binds atumor-specific antigen, a disease-associated antigen, or anextracellular matrix component. In some cases, where the first member ofthe specific binding pair is an antibody, the antibody specificallybinds a cell surface antigen, a soluble antigen, or an antigenimmobilized on an insoluble substrate. In some cases, where the firstmember of the specific binding pair is an antibody, the antibody is asingle-chain Fv. In some cases, the first member of the specific bindingpair is a nanobody, a single-domain antibody, a diabody, a triabody, ora minibody. In some cases, the first member of the specific binding pairis a non-antibody-based recognition scaffold. In some cases, where thefirst member of the specific binding pair is a non-antibody-basedrecognition scaffold, the non-antibody-based recognition scaffold is anavimer, a DARPin, an adnectin, an avimer, an affibody, an anticalin, oran affilin. In some cases, the first member of the specific binding pairis an antigen. In some cases, where the first member of the specificbinding pair is an antigen, the antigen is an endogenous antigen. Insome cases, where the first member of the specific binding pair is anantigen, the antigen is an exogenous antigen. In some cases, the firstmember of the specific binding pair is a ligand for a receptor. In somecases, the first member of the specific binding pair is a receptor. Insome cases, the first member of the specific binding pair is a cellularadhesion molecule (e.g., all or a portion of an extracellular region ofa cellular adhesion molecule). In some cases, the first member of thespecific binding pair comprises a first dimerization domain and whereinthe second member of the specific binding pair comprises a seconddimerization domain; for example, in some cases, binding of the firstdimerization domain to the second dimerization domain is induced by asmall molecule dimerization agent, and in other cases, binding of thefirst dimerization domain to the second dimerization domain is inducedby light. In some cases, the Notch receptor polypeptide comprises anamino acid sequence having at least 75% amino acid sequence identity toany one of the amino acid sequences depicted in FIGS. 2A-2G. In somecases, the Notch receptor polypeptide comprises an amino acid sequencehaving at least 85% amino acid sequence identity to any one of the aminoacid sequences depicted in FIGS. 2A-2G. In some cases, the Notchreceptor polypeptide comprises an amino acid sequence having at least90% amino acid sequence identity to any one of the amino acid sequencesdepicted in FIGS. 2A-2G. In some cases, the Notch receptor polypeptidecomprises an amino acid sequence having at least 95% amino acid sequenceidentity to any one of the amino acid sequences depicted in FIGS. 2A-2G.In some cases, the Notch receptor polypeptide comprises an amino acidsequence having at least 98% amino acid sequence identity to any one ofthe amino acid sequences depicted in FIGS. 2A-2G. In some cases, theNotch receptor polypeptide comprises an amino acid sequence having atleast 75% amino acid sequence identity to any one of the amino acidsequences depicted in FIG. 3. In some cases, the Notch receptorpolypeptide comprises an amino acid sequence having at least 85% aminoacid sequence identity to any one of the amino acid sequences depictedin FIG. 3. In some cases, the Notch receptor polypeptide comprises anamino acid sequence having at least 90% amino acid sequence identity toany one of the amino acid sequences depicted in FIG. 3. In some cases,the Notch receptor polypeptide comprises an amino acid sequence havingat least 95% amino acid sequence identity to any one of the amino acidsequences depicted in FIG. 3. In some cases, the Notch receptorpolypeptide comprises an amino acid sequence having at least 98% aminoacid sequence identity to any one of the amino acid sequences depictedin FIG. 3. In some cases, the Notch receptor polypeptide comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or 100% amino acid sequenceidentity to the following sequence:PPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLS (SEQ ID NO:1). In some cases, the Notch receptorpolypeptide comprises an amino acid sequence having at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or100% amino acid sequence identity to the following sequence:PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVG CGVLLS (SEQID NO:2). In some cases, the one or more ligand-inducible proteolyticcleavage sites are selected from S1, S2, and S3 proteolytic cleavagesites. In some cases, the S1 proteolytic cleavage site is a furin-likeprotease cleavage site comprising the amino acid sequenceArg-X-(Arg/Lys)-Arg, where X is any amino acid. In some cases, the S2proteolytic cleavage site ADAM-17-type protease cleavage site comprisingan Ala-Val dipeptide sequence. In some cases, the S3 proteolyticcleavage site is a γ-secretase cleavage site comprising a Gly-Valdipeptide sequence.

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a chimeric Notch receptor polypeptide as describedherein. The present disclosure provides a recombinant expression vectorcomprising a nucleotide sequence encoding a chimeric Notch receptorpolypeptide as described herein. The present disclosure provides a hostcell genetically modified with the nucleic acid, or the expressionvector. In some cases, the host cell is a eukaryotic cell. In somecases, the host cell is a mammalian cell. In some cases, the host cellis an immune cell, a neuron, an epithelial cell, and endothelial cell,or a stem cell. In some cases, the immune cell is a T cell, a B cell, amonocyte, a natural killer cell, a dendritic cell, or a macrophage. Insome cases, the host cell is genetically modified with a nucleic acidcomprising a nucleotide sequence encoding a chimeric antigen receptor(CAR), and wherein the intracellular domain of the chimeric polypeptideis a transcriptional activator. In some cases, the nucleotide sequenceencoding the CAR is operably linked to a transcriptional control elementthat is activated by the intracellular domain of the chimericpolypeptide.

The present disclosure provides a method of modulating an activity of acell that expresses a chimeric Notch receptor polypeptide of the presentdisclosure as described herein, the method comprising: contacting thecell with a second member of the specific binding pair, wherein bindingof the first member of the specific binding pair to the second member ofthe specific binding pair induces cleavage of the Notch receptorpolypeptide at the one or more ligand-inducible proteolytic cleavagesites, thereby releasing the intracellular domain, wherein release ofthe intracellular domain modulates the activity of the cell. In somecases, said contacting is carried out in vivo, ex vivo, or in vitro. Insome cases, the second member of the specific binding pair is on thesurface of a second cell, is immobilized on an insoluble substrate, ispresent in an extracellular matrix, is present in an artificial matrix,or is soluble. In some cases, release of the intracellular domainmodulates proliferation of the cell. In some cases, release of theintracellular domain modulates apoptosis in the cell. In some cases,release of the intracellular domain induces cell death by a mechanismother than apoptosis. In some cases, release of the intracellular domainmodulates gene expression in the cell through transcriptionalregulation, chromatin regulation, translation, trafficking orpost-translational processing. In some cases, release of theintracellular domain modulates differentiation of the cell. In somecases, release of the intracellular domain modulates migration of thecell. In some cases, release of the intracellular domain modulates theexpression and secretion of a molecule from the cell. In some cases,release of the intracellular domain modulates adhesion of the cell to asecond cell or to an extracellular matrix. In some cases, release of theintracellular domain induces de novo expression a gene product in thecell. In some cases, where release of the intracellular domain inducesde novo expression a gene product in the cell, the gene product is atranscriptional activator, a transcriptional repressor, a chimericantigen receptor, a second chimeric Notch receptor polypeptide, atranslation regulator, a cytokine, a hormone, a chemokine, or anantibody.

The present disclosure provides a method of modulating an activity of acell that expresses a chimeric Notch receptor polypeptide of the presentdisclosure as described herein, the method comprising: contacting thecell with a second member of the specific binding pair, where binding ofthe first member of the specific binding pair to the second member ofthe specific binding pair induces cleavage of the Notch receptorpolypeptide at the one or more ligand-inducible proteolytic cleavagesites, thereby releasing the intracellular domain, wherein theintracellular domain is a transcription factor that inducestranscription of a nucleic acid encoding an effector polypeptide thatmodulates the activity of the cell. In some cases, said contacting iscarried out in vivo, ex vivo, or in vitro. In some cases, the secondmember of the specific binding pair is on the surface of a second cell,is immobilized on an insoluble substrate, is present in an extracellularmatrix, is present in an artificial matrix, or is soluble. In somecases, the effector polypeptide is an apoptosis inducer, apoptosis ininhibitor, an activating immunoreceptor, an inhibiting immunoreceptor, atranscription activator, a transcription repressor, a cytokine, a growthfactor, a hormone, a receptor, an antibody, or a site-specific nuclease.

The present disclosure provides a method of modulating an activity of acell, the method comprising: contacting the cell with a second member ofa first specific binding pair, wherein the cell expresses: i) a firstchimeric Notch receptor polypeptide of the present disclosure asdescribed herein, comprising a first member of a first specific bindingpair; and ii) at least a second chimeric Notch receptor polypeptide ofthe present disclosure as described herein, comprising a first member ofa second specific binding pair, wherein the first and the secondspecific binding pairs are different from one another, wherein theintracellular domain of the first chimeric Notch receptor polypeptideprovides a first effector function; and the intracellular domain of thesecond chimeric Notch receptor polypeptide provides a second effectorfunction that is different from the first effector function, and whereinthe released first and the second intracellular domains modulateactivity of the cell. In some cases, said contacting is carried out invivo. In some cases, said contacting is carried out ex vivo. In somecases, said contacting is carried out in vitro.

The present disclosure provides a method of activating a T cell, themethod comprising: contacting a T cell as described herein (where the Tcell is genetically modified with one or more nucleic acids comprisingnucleotide sequences encoding: i) a chimeric Notch receptor polypeptideof the present disclosure; and ii) a CAR); with an immobilized antigen,wherein the extracellular domain of the chimeric Notch receptorpolypeptide comprises an antibody specific for a first antigen, andwherein said contacting results in release of the transcriptionalactivator, and production of the CAR in the cell, wherein the CARprovides for activation of the T cell following binding of a secondantigen.

The present disclosure provides a method of modulating an activity of acell, the method comprising: contacting the cell with a second member ofa first specific binding pair, wherein the cell expresses: i) a firstchimeric Notch receptor polypeptide of the present disclosure,comprising a first member of a first specific binding pair; and ii) atleast a second chimeric Notch receptor polypeptide of the presentdisclosure, comprising a first member of a second specific binding pair,wherein the first and the second specific binding pairs are differentfrom one another, wherein the nucleotide sequence encoding the secondchimeric Notch receptor is operably linked to a transcriptional controlelement that is activated or repressed by the intracellular domain ofthe first chimeric Notch receptor polypeptide. In some cases, saidcontacting is carried out in vivo. In some cases, said contacting iscarried out ex vivo. In some cases, said contacting is carried out invitro.

The present disclosure provides a method of activating a T cell, themethod comprising: contacting a T cell as described herein (where the Tcell is genetically modified with one or more nucleic acids comprisingnucleotide sequences encoding: i) a chimeric Notch receptor polypeptideof the present disclosure; and ii) a CAR, where the intracellular domainof the chimeric Notch receptor polypeptide is a transcriptionalactivator) with an immobilized antigen, wherein the extracellular domainof the chimeric polypeptide comprises an antibody specific for a firstantigen, and wherein said contacting results in release of thetranscriptional activator, and production of the CAR in the cell,wherein the CAR provides for activation of the T cell following bindingof a second antigen.

The present disclosure provides a method of modulating an activity of acell, the method comprising: contacting the cell with an antigen that isimmobilized on a surface, wherein the cell expresses a chimeric Notchreceptor polypeptide of the present disclosure, wherein the first memberof the specific binding pair binds the antigen, and wherein saidcontacting results in release of the intracellular domain and modulationof the activity of the cell. In some cases, the intracellular domain isa transcription factor that modulates differentiation of the cell.

The present disclosure provides method of locally modulating an activityof a cell, the method comprising: expressing in the cell abinding-triggered transcriptional switch comprising an extracellulardomain comprising a first member of a specific binding pair, abinding-transducer and an intracellular domain; and contacting the cellwith a second member of the specific binding pair, wherein binding ofthe first member of the specific binding pair to the second member ofthe specific binding pair induces the binding-transducer to transduce abinding signal to activate the intracellular domain, thereby producingan activated intracellular domain, wherein the activated intracellulardomain modulates an activity of the cell selected from the groupconsisting of: expression of a gene product of the cell, proliferationof the cell, apoptosis of the cell, non-apoptotic death of the cell,differentiation of the cell, dedifferentiation of the cell, migration ofthe cell, secretion of a molecule from the cell and cellular adhesion ofthe cell.

In some cases, the activated intracellular domain modulates expressionof an endogenous gene product of the cell.

In some cases, the endogenous gene product of the cell is selected fromthe group consisting of: a chemokine, a chemokine receptor, a cytokine,a cytokine receptor, a differentiation factor, a growth factor, a growthfactor receptor, a hormone, a metabolic enzyme, a proliferation inducer,a receptor, a small molecule 2^(nd) messenger synthesis enzyme, a T cellreceptor, a transcription activator, a transcription repressor, atranscriptional activator, a transcriptional repressor, a translationregulator, a translational activator, a translational repressor, anactivating immunoreceptor, an apoptosis inhibitor, an apoptosis inducer,an immunoactivator, an immunoinhibitor and an inhibiting immunoreceptor.

In some cases, the endogenous gene product of the cell is a secretedgene product. In some cases, the endogenous gene product of the cell isa surface expressed gene product. In some cases, the activatedintracellular domain simultaneously modulates expression of two or moreendogenous gene products of the cell. In some cases, the activatedintracellular domain modulates expression of a heterologous gene productof the cell.

In some cases, the heterologous gene product of the cell is selectedfrom the group consisting of: a chemokine, a chemokine receptor, achimeric antigen receptor, a cytokine, a cytokine receptor, adifferentiation factor, a growth factor, a growth factor receptor, ahormone, a metabolic enzyme, a pathogen derived protein, a proliferationinducer, a receptor, a RNA guided nuclease, a site-specific nuclease, asmall molecule 2nd messenger synthesis enzyme, a T cell receptor, atoxin derived protein, a transcription activator, a transcriptionrepressor, a transcriptional activator, a transcriptional repressor, atranslation regulator, a translational activator, a translationalrepressor, an activating immunoreceptor, an antibody, an apoptosis ininhibitor, an apoptosis inducer, an engineered T cell receptor, animmunoactivator, an immunoinhibitor, an inhibiting immunoreceptor, anRNA guided DNA binding protein and a second binding-triggeredtranscriptional switch.

In some instances, the heterologous gene product of the cell is anantibody selected from the group consisting of: 806, 9E10, 3F8, 81C6,8H9, Abagovomab, Abatacept, Abciximab, Abituzumab, Abrilumab, Actoxumab,Adalimumab, Adecatumumab, Aducanumab, Afelimomab, Afutuzumab, Alacizumabpegol, ALD518, Alefacept, Alemtuzumab, Alirocumab, Altumomab pentetate,Amatuximab, AMG 102, Anatumomab mafenatox, Anetumab ravtansine,Anifrolumab, Anrukinzumab, Apolizumab, Arcitumomab, Ascrinvacumab,Aselizumab, Atacicept, Atezolizumab, Atinumab, Atlizumab/tocilizumab,Atorolimumab, AVE1642, Bapineuzumab, Basiliximab, Bavituximab,Bectumomab, Begelomab, Belimumab, Benralizumab, Bertilimumab,Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab,Bimekizumab, Bivatuzumab mertansine, Blinatumomab, Blosozumab,BMS-936559, Bococizumab, Brentuximab vedotin, Briakinumab, Brodalumab,Brolucizumab, Brontictuzumab, Canakinumab, Cantuzumab mertansine,Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab,Catumaxomab, cBR96-doxorubicin immunoconjugate, CC49, CDP791,Cedelizumab, Certolizumab pegol, Cetuximab, cG250, Ch.14.18, Citatuzumabbogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumabtetraxetan, Codrituzumab, Coltuximab ravtansine, Conatumumab,Concizumab, CP 751871, CR6261, Crenezumab, CS-1008, Dacetuzumab,Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab,Demcizumab, Denintuzumab mafodotin, Denosumab, Derlotuximab biotin,Detumomab, Dinutuximab, Diridavumab, Dorlimomab aritox, Drozitumab,Duligotumab, Dupilumab, Durvalumab, Dusigitumab, Ecromeximab,Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab,Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab, Emibetuzumab,Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab,Enokizumab, Enoticumab, Ensituximab, Epitumomab cituxetan, Epratuzumab,Erlizumab, Ertumaxomab, Etanercept, Etaracizumab, Etrolizumab,Evinacumab, Evolocumab, Exbivirumab, F19, Fanolesomab, Faralimomab,Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Ficlatuzumab,Figitumumab, Firivumab, Flanvotumab, Fletikumab, Fontolizumab,Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab,Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin,Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab,Gomiliximab, Guselkumab, HGS-ETR2, hu3S193, huA33, Ibalizumab,Ibritumomab tiuxetan, Icrucumab, Idarucizumab, IGN101, IgN311, Igovomab,IIIA4, IM-2C6, IMAB362, Imalumab, IMC-A12, Imciromab, Imgatuzumab,Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Infliximab,Inolimomab, Inotuzumab ozogamicin, Intetumumab, Ipilimumab, Iratumumab,Isatuximab, Itolizumab, Ixekizumab, J591, KB004, Keliximab, KW-2871,Labetuzumab, Lambrolizumab, Lampalizumab, Lebrikizumab, Lemalesomab,Lenzilumab, Lerdelimumab, Lexatumumab, Libivirumab, Lifastuzumabvedotin, Ligelizumab, Lilotomab satetraxetan, Lintuzumab, Lirilumab,Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, Lucatumumab,Lulizumab pegol, Lumiliximab, Lumretuzumab, Mapatumumab, Margetuximab,Maslimomab, Matuzumab, Mavrilimumab, MEDI4736, Mepolizumab, Metelimumab,METMAB, Milatuzumab, Minretumomab, Mirvetuximab soravtansine, Mitumomab,MK-0646, MK-3475, MM-121, Mogamulizumab, MORAb-003, Morolimumab,Motavizumab, MOv18, Moxetumomab pasudotox, MPDL33280A, Muromonab-CD3,Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Narnatumab,Natalizumab, Nebacumab, Necitumumab, Nemolizumab, Nerelimomab,Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan,Obiltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab,Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab,Ontuxizumab, Opicinumab, Oportuzumab monatox, Oregovomab, Orticumab,Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab,Pagibaximab, Palivizumab, Panitumumab, Pankomab, Panobacumab,Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab,Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab,Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumabvedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140,Quilizumab, R1507, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab,Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab, Regavirumab,Reslizumab, Rilotumumab, Rinucumab, Rituximab, Robatumumab, Roledumab,Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Sacituzumabgovitecan, Samalizumab, Sarilumab, Satumomab pendetide, SCH 900105,Secukinumab, Seribantumab, Setoxaximab, Sevirumab, SGN-CD19A, SGN-CD33A,Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab,Sirukumab, Sofituzumab vedotin, Solanezumab, Solitomab, Sonepcizumab,Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumabtetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox,Tarextumab, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab,Teplizumab, Teprotumumab, Tesidolumab, Tetulomab, TGN1412,Ticilimumab/tremelimumab, Tigatuzumab, Tildrakizumab, TNX-650,Tocilizumab, Toralizumab, Tosatoxumab, Tositumomab, Tovetumab,Tralokinumab, Trastuzumab, TRBS07, Tregalizumab, Tremelimumab,Trevogrumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab,Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab, Vandortuzumab vedotin,Vantictumab, Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab,Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab,Volociximab, Vorsetuzumab mafodotin, Votumumab, Zalutumumab,Zanolimumab, Zatuximab, Ziralimumab and Zolimomab aritox.

In some cases, the heterologous gene product of the cell is a secretedgene product. In some cases, the heterologous gene product of the cellis a surface expressed gene product. In some cases, the activatedintracellular domain simultaneously modulates expression of two or moreheterologous gene products of the cell. In some cases, the contacting iscarried out in vivo, ex vivo, or in vitro.

In some cases, the second member of the specific binding pair is on thesurface of a second cell, is immobilized on an insoluble substrate, ispresent in an extracellular matrix, is present in an artificial matrix,or is soluble. In some cases, the intracellular the transcription factordirectly modulates differentiation of the cell. In some cases, thetranscription factor indirectly modulates differentiation of the cell bymodulating the expression of a second transcription factor.

In some cases, the cell is an immune cell and the activity of the cellis differentiation of the immune cell. In some cases, the cell is animmune cell, the intracellular domain is a transcription factor thatmodulates differentiation of the cell and the activity of the cell isdifferentiation of the immune cell. In some cases, the transcriptionfactor directly modulates differentiation of the immune cell. In somecases, the transcription factor indirectly modulates differentiation ofthe immune cell by modulating the expression of a second transcriptionfactor.

In some cases, the cell is a stem cell and the activity of the cell isdifferentiation of the stem cell. In some cases, the cell is aprogenitor or precursor cell and the activity of the cell isdifferentiation of the progenitor or precursor cell.

In some cases, activation of the intracellular domain modulatesexpression of an endogenous gene of the cell through transcriptionalregulation, chromatin regulation, translation, trafficking orpost-translational processing. In some cases, activation of theintracellular domain modulates cellular adhesion of the cell to a secondcell or to an extracellular matrix.

In some cases, the binding-transducer comprises a ligand-inducibleproteolytic cleavage site, wherein binding of the first member of thespecific binding pair to the second member of the specific binding pairinduces cleavage of the binding-transducer at the ligand-inducibleproteolytic cleave site, thereby transducing the binding signal andactivating the intracellular domain by proteolytically releasing theintracellular domain.

The present disclosure provides a method of modulating an activity of acell, the method comprising: contacting the cell with a second member ofa first specific binding pair and a second member of a second specificbinding pair, wherein the cell expresses: i) a first binding-triggeredtranscriptional switch comprising an extracellular domain comprising afirst member of the first specific binding pair, a binding-transducerand an intracellular domain; and ii) at least a second binding-triggeredtranscriptional switch comprising an extracellular domain comprising thefirst member of a second specific binding pair, a binding-transducer andan intracellular domain; wherein the intracellular domain of the firstbinding-triggered transcriptional switch provides a first effectorfunction and the intracellular domain of the second binding-triggeredtranscriptional switch provides a second effector function that isdifferent from the first effector function when binding of the first andsecond members of the first and second specific binding pairs inducesthe binding-transducers to transduce binding signals to activate thefirst and second intracellular domains.

In some cases, the effector function of the intracellular domain of thefirst binding-triggered transcriptional switch modulates expression of agene product of the cell.

In some cases, the gene product of the cell is an endogenous geneproduct of the cell. In some cases, the gene product of the cell is aheterologous gene product of the cell. In some cases, the gene productof the cell is a gene product of the cell is selected from the groupconsisting of: a chemokine, a chemokine receptor, a cytokine, a cytokinereceptor, a differentiation factor, a growth factor, a growth factorreceptor, a hormone, a metabolic enzyme, a proliferation inducer, areceptor, a small molecule 2^(nd) messenger synthesis enzyme, a T cellreceptor, a transcription activator, a transcription repressor, atranscriptional activator, a transcriptional repressor, a translationregulator, a translational activator, a translational repressor, anactivating immunoreceptor, an apoptosis in inhibitor, an apoptosisinducer, an immunoactivator, an immunoinhibitor and an inhibitingimmunoreceptor.

In some cases, the effector function of the intracellular domain of thesecond binding-triggered transcriptional switch modulates expression ofa gene product of the cell. In some cases, the gene product of the cellis an endogenous gene product of the cell. In some cases, the geneproduct of the cell is a heterologous gene product of the cell.

In some cases, the gene product of the cell is selected from the groupconsisting of: a chemokine, a chemokine receptor, a cytokine, a cytokinereceptor, a differentiation factor, a growth factor, a growth factorreceptor, a hormone, a metabolic enzyme, a proliferation inducer, areceptor, a small molecule 2^(nd) messenger synthesis enzyme, a T cellreceptor, a transcription activator, a transcription repressor, atranscriptional activator, a transcriptional repressor, a translationregulator, a translational activator, a translational repressor, anactivating immunoreceptor, an apoptosis in inhibitor, an apoptosisinducer, an immunoactivator, an immunoinhibitor and an inhibitingimmunoreceptor.

In some cases, at least one of the binding-transducers of the first andsecond binding-triggered transcriptional switches comprises aligand-inducible proteolytic cleavage site, wherein binding of the firstand second members of the respective specific binding pair inducescleavage of the binding-transducer at the ligand-inducible proteolyticcleave site, thereby transducing the binding signal and activating therespective intracellular domain by proteolytically releasing theintracellular domain.

In some cases, the binding-transducers of the first and secondbinding-triggered transcriptional switches both comprise aligand-inducible proteolytic cleavage site.

In some instances, the method further includes contacting the cell witha soluble inhibitor molecule that competitively inhibits the binding ofthe first member of the specific binding pair to the second member ofthe specific binding pair, thereby preventing induction of thebinding-transducer to transduce a binding signal to activate theintracellular domain, wherein contacting the cell with the solubleinhibitor molecule comprises applying or administering the solubleinhibitor molecule to first cell and/or placing the cell in the presenceof a second cell that expresses the soluble inhibitor molecule. In someinstances, the second cell constitutively expresses the solubleinhibitor molecule. In some instances, the second cell conditionallyexpresses the soluble inhibitor molecule.

The present disclosure provides a method of modulating an activity of acell, the method comprising: contacting the cell with a second member ofa first specific binding pair, wherein the cell expresses: i) a firstbinding-triggered transcriptional switch comprising an extracellulardomain comprising a first member of the first specific binding pair, abinding-transducer and an intracellular domain; and ii) at least asecond binding-triggered transcriptional switch comprising anextracellular domain comprising the first member of a second specificbinding pair, a binding-transducer and an intracellular domain, whereinthe nucleotide sequence encoding the second binding-triggeredtranscriptional switch is operably linked to a transcriptional controlelement that is activated or repressed by the intracellular domain ofthe first binding-triggered transcriptional switch.

In some cases, the contacting is carried out in vivo, ex vivo, or invitro. In some cases, the second member of the first specific bindingpair is on the surface of a second cell, is immobilized on an insolublesubstrate, is present in an extracellular matrix, is present in anartificial matrix, or is soluble.

In some cases, activation of the intracellular domain of the secondbinding-triggered transcriptional switch modulates an activity of thecell selected from the group consisting of: expression of a gene productof the cell, proliferation of the cell, apoptosis of the cell,non-apoptotic death of the cell, differentiation of the cell,dedifferentiation of the cell, migration of the cell, secretion of amolecule from the cell and cellular adhesion of the cell.

In some cases, the activity of the cell is expression of a gene productof the cell. In some cases, the gene product of the cell is a geneproduct of the cell is selected from the group consisting of: achemokine, a chemokine receptor, a cytokine, a cytokine receptor, adifferentiation factor, a growth factor, a growth factor receptor, ahormone, a metabolic enzyme, a proliferation inducer, a receptor, asmall molecule 2^(nd) messenger synthesis enzyme, a T cell receptor, atranscription activator, a transcription repressor, a transcriptionalactivator, a transcriptional repressor, a translation regulator, atranslational activator, a translational repressor, an activatingimmunoreceptor, an apoptosis in inhibitor, an apoptosis inducer, animmunoactivator, an immunoinhibitor and an inhibiting immunoreceptor.

In some cases, at least one of the binding-transducers of the first andsecond binding-triggered transcriptional switches comprises aligand-inducible proteolytic cleavage site, wherein binding of the firstand second members of the respective specific binding pair inducescleavage of the binding-transducer at the ligand-inducible proteolyticcleave site, thereby transducing the binding signal and activating therespective intracellular domain by proteolytically releasing theintracellular domain.

The present disclosure provides a method of tracking cell-cell contacts,the method comprising: expressing in each cell of a first plurality ofcells an binding-triggered transcriptional switch comprising anextracellular domain comprising a first member of a specific bindingpair, a binding-transducer and an intracellular domain; expressing ineach cell of a second plurality of cells a second member of the specificbinding pair; and contacting the first plurality of cells with thesecond plurality of cells, wherein binding of the first member of thespecific binding pair to the second member of the specific binding pairinduces the binding-transducer to transduce a binding signal of thebinding-triggered transcriptional switch, thereby activating theintracellular domain, wherein activation of the intracellular domaininduces expression of a detectable reporter sufficient to trackcell-cell contacts in space, in time or a combination thereof.

In some cases, the first plurality of cells, the second plurality ofcells or both are neurons. In some cases, the binding-transducercomprises a ligand-inducible proteolytic cleavage site, wherein bindingof the first member of the specific binding pair to the second member ofthe specific binding pair induces cleavage of the binding-transducer atthe ligand-inducible proteolytic cleave site, thereby transducing thebinding signal and activating the intracellular domain byproteolytically releasing the intracellular domain.

In some cases, the binding-triggered transcriptional switch, includingthose described above and herein is a SynNotch polypeptide.

The present disclosure also provides a localized cell activation system,the system comprising: a cell comprising: an expressed binding-triggeredtranscriptional switch comprising an extracellular domain comprising afirst member of a first specific binding pair, a binding-transducer andan intracellular domain; and a nucleic acid, operably linked to atranscriptional control element that is induced by the intracellulardomain of the first binding-triggered transcriptional switch, encoding abinding-triggered activating polypeptide comprising a first member of asecond specific binding pair; wherein upon contact with the secondmember of the first specific binding pair the binding-triggeredactivating polypeptide is expressed and upon contact with the secondmember of the second specific binding pair the binding-triggeredactivating polypeptide activates the cell.

In some cases, the cell is selected from the group consisting of: animmune cell, a progenitor or precursor cell, a stem cell and a neuron.In some cases, the cell is an immune cell, the binding-triggeredtranscriptional switch is an antigen triggered transcriptional switchand the binding-triggered activating polypeptide is an antigen triggeredactivating polypeptide, wherein upon contact with the second member ofthe first specific binding pair the antigen triggered activatingpolypeptide is expressed and upon contact with the second member of thesecond specific binding pair the antigen triggered activatingpolypeptide activates the immune cell to recognize target cellsexpressing the first member of the second specific binding pair.

In some cases, the antigen triggered activating polypeptide is achimeric antigen receptor or a variant thereof. In some cases, theantigen triggered activating polypeptide is an engineered T cellreceptor or a variant thereof. In some cases, the expressedbinding-triggered transcriptional switch is a SynNotch polypeptide.

The present disclosure provides a method of locally modulating anactivity of a cell, the method comprising: expressing in a first cell abinding-triggered transcriptional switch comprising abinding-transducer, an intracellular domain and a first extracellulardomain comprising a first adaptor binding domain that specifically bindsa first epitope on a soluble adaptor molecule; contacting the first cellwith: i) a second cell that expresses a second extracellular domaincomprising a second adaptor binding domain that specifically binds asecond epitope on the soluble adaptor molecule; and ii) an effectiveconcentration of the soluble adaptor molecule, wherein binding of thefirst adaptor binding domain and the second adaptor binding domain tothe adaptor molecule induces the binding-transducer to transduce abinding signal to activate the intracellular domain, thereby producingan activated intracellular domain, wherein the activated intracellulardomain modulates an activity of the first cell that is selected from thegroup consisting of: expression of a gene product of the cell,proliferation of the cell, apoptosis of the cell, non-apoptotic death ofthe cell, differentiation of the cell, dedifferentiation of the cell,migration of the cell, secretion of a molecule from the cell andcellular adhesion of the cell. In some instances, the contactingcomprises applying the soluble adaptor molecule to the cells in vitro orex vivo or administering the soluble adaptor molecule to the cells invivo. In some instances, contacting the first cell with an effectiveconcentration of the soluble adaptor molecule comprises placing thefirst cell in the presence of a third cell that expresses the adaptormolecule, wherein the third cell constitutively or conditionallyexpresses the adaptor molecule. In some instances, the firstextracellular domain and the soluble adaptor molecule are first andsecond members of a specific binding pair. In some instances, the secondextracellular domain and the soluble adaptor molecule are first andsecond members of a specific binding pair. In some instances, the firstextracellular domain and second extracellular domain are antibodies ornanobodies. In some instances, the intracellular domain is atranscription factor. In some instances, the activated intracellulardomain modulates expression of an endogenous or heterologous geneproduct of the first cell. In some instances, the binding-transducercomprises a ligand-inducible proteolytic cleavage site, wherein bindingof the first extracellular domain and the second extracellular domain tothe soluble adaptor molecule induces cleavage of the binding-transducerat the ligand-inducible proteolytic cleave site, thereby transducing thebinding signal and activating the intracellular domain byproteolytically releasing the intracellular domain.

The present disclosure provides a host cell comprising: a nucleic acidencoding a first binding-triggered transcriptional switch responsive toa first antigen; a first promoter that is responsive to the firstbinding-triggered transcriptional switch and is operably linked to anucleic acid encoding a CAR comprising an extracellular domain thatspecifically binds to a first member of a specific binding pair; anucleic acid encoding a second binding-triggered transcriptional switchresponsive to a second antigen; and a second promoter that is responsiveto the second binding-triggered transcriptional switch and operablylinked to nucleic acid encoding an intracellular CAR inhibitory domain,wherein in the presence of the second antigen the intracellular CARinhibitory domain is expressed inhibiting activation of the cell by theCAR and in the presence of the first antigen but not the second antigenthe CAR is expressed and activatable by the second member of thespecific binding pair.

The present disclosure provides a host cell comprising: a nucleic acidencoding a first binding-triggered transcriptional switch responsive toa first antigen; a first promoter that is responsive to the firstbinding-triggered transcriptional switch and is operably linked to anucleic acid encoding a first portion of a CAR comprising anextracellular domain that specifically binds to a first member of aspecific binding pair; a nucleic acid encoding a secondbinding-triggered transcriptional switch responsive to a second antigen;and a second promoter that is responsive to the second binding-triggeredtranscriptional switch and operably linked to nucleic acid encoding asecond portion of a CAR comprising an intracellular signaling domain,wherein in the presence of the first antigen and second antigen thefirst and second portions of the CAR are expressed and the CAR isactivatable by the second member of the specific binding pair. In someinstances, the cell further comprises a nucleic acid encoding a thirdbinding-triggered transcriptional switch responsive to a third antigen;and a third promoter that is responsive to the third binding-triggeredtranscriptional switch and is operably linked to a nucleic acid encodingan intracellular CAR inhibitory domain, wherein in the presence of thethird antigen the intracellular CAR inhibitory domain is expressedinhibiting activation of the cell by the CAR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of a Notch receptor polypeptide.

FIGS. 2A-2G provide amino acid sequences of Notch receptor polypeptidesof various species (SEQ ID NOs:131-137).

FIG. 3 provides an amino acid sequence alignment of a portion of Notchreceptor polypeptides of various mammalian species (mouse—SEQ ID NO:138;human—SEQ ID NO:139; cow—SEQ ID NO:140).

FIGS. 4A-4G provide schematic depictions of exemplary chimeric Notchreceptor polypeptides of the present disclosure.

FIG. 5 provides a schematic depiction of direct control of effectorfunction, using a chimeric Notch receptor polypeptide of the presentdisclosure.

FIG. 6 provides a schematic depiction of an example of direct control ofeffector function, using a chimeric Notch receptor polypeptide of thepresent disclosure.

FIG. 7 provides a schematic depiction of indirect control of effectorfunction, using a chimeric Notch receptor polypeptide of the presentdisclosure.

FIG. 8 provides a schematic depiction of an example of indirect controlof effector function, using a chimeric Notch receptor polypeptide of thepresent disclosure.

FIGS. 9A and 9B provide schematic depictions of use of multiple chimericNotch receptor polypeptides in parallel.

FIG. 10 provides a schematic depiction of use of multiple chimeric Notchreceptor polypeptides in series.

FIG. 11 provides a schematic depiction of use of a chimeric Notchreceptor polypeptide and a chimeric antigen receptor (CAR) in series.

FIG. 12 provides a schematic depiction of use of a chimeric Notchreceptor polypeptide in two or more cells, showing multi-cellcooperativity.

FIG. 13 provides a schematic depiction of use of a chimeric Notchreceptor polypeptide in a multicellular environment.

FIG. 14 provides a schematic depiction of use of multiple receptorcircuits with two or more cells.

FIG. 15 provides a schematic depiction of localized/targeted productionof biologics in response to specific extracellular structures.

FIGS. 16A-16C depict examples of Notch receptor polypeptides (SEQ IDNOs:141-143).

FIGS. 17-29 provide amino acid sequences of exemplary chimeric Notchreceptor polypeptides (FIG. 17A-C—SEQ ID NOs:144-146; FIG. 18—SEQ IDNO:147; FIG. 19A-B—SEQ ID NOs:148-149; FIG. 20—SEQ ID NOs:150-153; FIG.21—SEQ ID NO:154; FIG. 22—SEQ ID NO:155; FIG. 23—SEQ ID NO:156; FIG.24—SEQ ID NO:157; FIG. 25—SEQ ID NO:158; FIG. 26—SEQ ID NO:159; FIG.27—SEQ ID NO:160; FIG. 28—SEQ ID NO:161; FIG. 29—SEQ ID NO:162).

FIGS. 30A and 30B depict representative results for the Chimeric Notchwith anti-CD19 in the TRE reporter line.

FIGS. 31A and 31B depict representative results for the Chimeric Notchwith anti-mesothelin in the TRE reporter line.

FIGS. 32A and 32B depict representative results for the Chimeric Notchanti-CD19 in the UAS reporter line.

FIGS. 33A and 33B depict depicts results with SV40/UAS reporter cellstransduced with the anti-CD19 Chimeric Notch in which the intracellulardomain is a fusion of the Gal4 DNA-binding domain with thetranscriptional repressor domain KRAB.

FIG. 34 depicts use of chimeric Notch receptor polypeptides in a cascadeof signaling relay.

FIG. 35A-35C depicts the effect of a chimeric Notch receptor polypeptideon Chimeric Antigen Receptor (CAR) expression and T cell activation tocancer cells.

FIG. 36 provides an amino acid sequence of a Cas9 polypeptide (SEQ IDNO:163).

FIGS. 37-83 provide amino acid sequences of exemplary transcriptionactivators and repressors (FIG. 37—SEQ ID NO:164; FIG. 38—SEQ ID NO:165;FIG. 39—SEQ ID NO:165; FIG. 40—SEQ ID NO:166; FIG. 41—SEQ ID NO:167;FIG. 42—SEQ ID NO:169; FIG. 43—SEQ ID NO:170; FIG. 44—SEQ ID NO:171;FIG. 45—SEQ ID NO:172; FIG. 46—SEQ ID NO:173; FIG. 47—SEQ ID NO:174;FIG. 48—SEQ ID NO:175; FIG. 49—SEQ ID NO:176; FIG. 50—SEQ ID NO:177;FIG. 51—SEQ ID NO:178; FIG. 52—SEQ ID NO:179; FIG. 53—SEQ ID NO:180;FIG. 54—SEQ ID NO:181; FIG. 55—SEQ ID NO:182; FIG. 56—SEQ ID NO:183;FIG. 57—SEQ ID NO:184; FIG. 58—SEQ ID NO:185; FIG. 59—SEQ ID NO:186;FIG. 60—SEQ ID NO:187; FIG. 61—SEQ ID NO:188; FIG. 62—SEQ ID NO:189;FIG. 63—SEQ ID NO:190; FIG. 64—SEQ ID NO:191; FIG. 65—SEQ ID NO:192;FIG. 66—SEQ ID NO:193; FIG. 67—SEQ ID NO:194; FIG. 68—SEQ ID NO:195;FIG. 69—SEQ ID NO:196; FIG. 70—SEQ ID NO:197; FIG. 71—SEQ ID NO:198;FIG. 72—SEQ ID NO:199; FIG. 73—SEQ ID NO:200; FIG. 74—SEQ ID NO:201;FIG. 75—SEQ ID NO:202; FIG. 76—SEQ ID NO:203; FIG. 77—SEQ ID NO:204;FIG. 78—SEQ ID NO:205; FIG. 79—SEQ ID NO:206; FIG. 80—SEQ ID NO:207;FIG. 81—SEQ ID NO:208; FIG. 82—SEQ ID NO:209; FIG. 83—SEQ ID NO:210).

FIGS. 84A and 84B depict the effect of a γ-secretase inhibitor onactivation of a chimeric Notch receptor polypeptide.

FIGS. 85A and 85B depict exemplary modular configurations of synNotchreceptors.

FIGS. 86A-86C demonstrate that SynNotch receptors can be used to programcontact dependent transcriptional regulation.

FIGS. 87A-87D provides additional data related to FIGS. 85A and 85B.

FIGS. 88A-88C provides additional data related to FIGS. 86A-C.

FIGS. 89A and 89B demonstrate that SynNotch receptors function indiverse cell types.

FIGS. 90A-90C provide additional data related to FIGS. 89A and 89B.

FIGS. 91A-91D demonstrate that SynNotch receptors yield spatial controlof diverse cellular behaviors.

FIGS. 92A-92C provide additional data related to FIGS. 91A-D.

FIGS. 93A-93C demonstrate that SynNotch receptors are orthogonal to oneanother and can be used for combinatorial regulation.

FIGS. 94A-94C demonstrate that multiple synNotch receptors can be usedto generate multi-layered self-organizing epithelial patterns.

FIGS. 95A-95C show that the modularity of synNotch receptors expandssensing/response engineering of mammalian cells.

FIGS. 96A-96C demonstrate the potential to engineer customizedtherapeutic T cell responses using synNotch Receptors.

FIGS. 97A-97F show that synNotch receptors can drive antigen-inducedtranscription in CD4+ and CD8+ human primary T lymphocytes.

FIGS. 98A-98F demonstrate that synNotch receptors can driveantigen-induced custom cytokine programs.

FIGS. 99A-99E demonstrates that synNotch receptors can driveantigen-dependent skewing of T cell differentiation to the anti-tumorTh1 fate.

FIGS. 100A-100E demonstrate custom T cell delivery of non-nativetherapeutic—synNotch driven TRAIL production.

FIGS. 101A-101C demonstrate in vivo local expression of cytokines atsolid tumors via a synNotch receptor engineered T cell.

FIGS. 102A-102B demonstrate that synNotch receptors are versatileregulators that allow T cells to monitor and selectively modulate theirmicroenvironment.

FIGS. 103A-103F provide supplemental data related to FIGS. 97A-97F.

FIGS. 104A-104I provide supplemental data related to FIGS. 98A-98F.

FIGS. 105A-105G provide supplemental data related to FIGS. 99A-99E.

FIGS. 106A-106H provide supplemental data related to FIGS. 100A-100E.

FIGS. 107A-107E provide supplemental data related to FIGS. 101A-101C.

FIGS. 108A-108D provide embodiments of synNotch receptors forcombinatorial antigen sensing in T cells.

FIGS. 109A-109D demonstrate synNotch-Gated CAR expression—combinatorialantigen requirement for Jurkat T cell activation.

FIGS. 110A-110F demonstrate synNotch Gated CAR expression in humanprimary T cells—combinatorial antigen control over therapeutic T cellactivation and tumor killing.

FIGS. 111A-111C show synNotch receptors driving tumor localized CARexpression in vivo.

FIGS. 112A-112D show selective combinatorial antigen tumor killing invivo by synNotch gated CAR expression.

FIGS. 113A-113C show that synNotch receptors control and localize CAR Tcell response for precision immunotherapy.

FIGS. 114A-114D provide supplemental data related to FIGS. 109A-109D.

FIGS. 115A-115I provide supplemental data related to FIGS. 110A-110F.

FIGS. 116A-116C provide supplemental data related to FIGS. 111A-111C.

FIGS. 117A-117E provide supplemental data related to FIGS. 112A-112D.

FIG. 118 demonstrates the induction of Foxp3 expression in human T cellsusing synNotch.

FIG. 119 provides a general antibody construct design for SynNotchcontrolled antibody secretion from human T cells.

FIG. 120 provides a schematic representation of an in vitro assay usedto test SynNotch induced antibody secretion.

FIG. 121 provides a schematic representation of a cell surface “SandwichELISA” flow cytometry assay.

FIG. 122 demonstrates that SynNotch T cells can be induced to produce aheterologous antibody in response to antigen stimulation.

FIG. 123 provides a schematic representation of a split SynNotchsignaling system.

FIG. 124 demonstrates receiver cell activation at various concentrationsof soluble adapter molecule according to an embodiment of a splitSynNotch signaling system of the instant disclosure.

FIG. 125 provides a schematic representation of a three cell splitSynNotch signaling system according to an embodiment of the instantdisclosure.

FIG. 126 demonstrates SynNotch receiver cell activation in a three cellsplit SynNotch signaling system according to an embodiment of theinstant disclosure.

FIG. 127 provides a schematic representation of a three cell splitSynNotch inhibitory signaling system according to an embodiment of theinstant disclosure.

FIG. 128 demonstrates inhibition of SynNotch receiver cell activation ina three cell split SynNotch inhibitory signaling system according to anembodiment of the instant disclosure.

FIGS. 129A-129F provide schematic representations of particularembodiments of split CAR systems as described herein (FIG. 129Fidentifies the elements of the schematics represented in FIGS.129A-129E).

FIG. 130 depicts one embodiment of a two antigen gated split CARcircuit.

FIG. 131 depicts an additional embodiment of a two antigen gated splitCAR circuit.

FIG. 132 provides a schematic representation of an embodiment of a threeinput gated circuit.

FIG. 133 provides a diagram of one configuration of a three antigengated SynNotch, split CAR circuit.

FIG. 134 provides a schematic representation of an embodiment of a fourinput gated circuit.

FIG. 135 provides a schematic representation of an embodiment of a fiveinput gated circuit.

FIG. 136 provides a schematic representation of one embodiment of athree input AND+NOT gate of the present disclosure.

FIG. 137 provides a schematic representation of one embodiment of amulti-input gate with split transcription factor AND functionality anddominant negative NOT functionality.

FIG. 138 provides a schematic representation of one embodiment of amulti-input gated CAR T cell activation circuit.

FIG. 139 provides the results of an analysis, similar to the analysisperformed in FIG. 122, of SynNotch CD4 T cells modified to conditionallysecrete pembrolizumab.

FIG. 140 provides the results of an analysis, similar to the analysisperformed in FIG. 122, of SynNotch E6-1 Jurkat cells modified to secretepembrolizumab.

FIG. 141 provides the results of an analysis, similar to the analysisperformed in FIG. 122, of SynNotch E6-1 Jurkat cells modified to secretepembrolizumab using an alternative construct.

FIG. 142 provides the results of an analysis, similar to the analysisperformed in FIG. 122, of SynNotch CD4 T cells modified to secreteTremelimumab.

FIG. 143 provides the results of an analysis, similar to the analysisperformed in FIG. 122, of SynNotch E6-1 Jurkat cells modified to secreteTremelimumab.

DEFINITIONS

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includes, butis not limited to, single-, double-, or multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. For instance, a promoter is operably linked to a codingsequence if the promoter affects its transcription or expression.

A “vector” or “expression vector” is a replicon, such as plasmid, phage,virus, or cosmid, to which another DNA segment, i.e. an “insert”, may beattached so as to bring about the replication of the attached segment ina cell.

“Heterologous,” as used herein, means a nucleotide or polypeptidesequence that is not found in the native (e.g., naturally-occurring)nucleic acid or protein, respectively.

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies that retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,single-chain antibodies (scAb), single domain antibodies (dAb), singledomain heavy chain antibodies, a single domain light chain antibodies,nanobodies, bi-specific antibodies, multi-specific antibodies, andfusion proteins comprising an antigen-binding (also referred to hereinas antigen binding) portion of an antibody and a non-antibody protein.The antibodies can be detectably labeled, e.g., with a radioisotope, anenzyme that generates a detectable product, a fluorescent protein, andthe like. The antibodies can be further conjugated to other moieties,such as members of specific binding pairs, e.g., biotin (member ofbiotin-avidin specific binding pair), and the like. The antibodies canalso be bound to a solid support, including, but not limited to,polystyrene plates or beads, and the like. Also encompassed by the termare Fab′, Fv, F(ab′)₂, and or other antibody fragments that retainspecific binding to antigen, and monoclonal antibodies. As used herein,a monoclonal antibody is an antibody produced by a group of identicalcells, all of which were produced from a single cell by repetitivecellular replication. That is, the clone of cells only produces a singleantibody species. While a monoclonal antibody can be produced usinghybridoma production technology, other production methods known to thoseskilled in the art can also be used (e.g., antibodies derived fromantibody phage display libraries). An antibody can be monovalent orbivalent. An antibody can be an Ig monomer, which is a “Y-shaped”molecule that consists of four polypeptide chains: two heavy chains andtwo light chains connected by disulfide bonds.

The term “humanized immunoglobulin” as used herein refers to animmunoglobulin comprising portions of immunoglobulins of differentorigin, wherein at least one portion comprises amino acid sequences ofhuman origin. For example, the humanized antibody can comprise portionsderived from an immunoglobulin of nonhuman origin with the requisitespecificity, such as a mouse, and from immunoglobulin sequences of humanorigin (e.g., chimeric immunoglobulin), joined together chemically byconventional techniques (e.g., synthetic) or prepared as a contiguouspolypeptide using genetic engineering techniques (e.g., DNA encoding theprotein portions of the chimeric antibody can be expressed to produce acontiguous polypeptide chain). Another example of a humanizedimmunoglobulin is an immunoglobulin containing one or moreimmunoglobulin chains comprising a complementarity-determining region(CDR) derived from an antibody of nonhuman origin and a framework regionderived from a light and/or heavy chain of human origin (e.g.,CDR-grafted antibodies with or without framework changes). Chimeric orCDR-grafted single chain antibodies are also encompassed by the termhumanized immunoglobulin. See, e.g., Cabilly et al., U.S. Pat. No.4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss etal., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al.,European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539;Winter, European Patent No. 0,239,400 B1; Padlan, E. A. et al., EuropeanPatent Application No. 0,519,596 A1. See also, Ladner et al., U.S. Pat.No. 4,946,778; Huston, U.S. Pat. No. 5,476,786; and Bird, R. E. et al.,Science, 242: 423-426 (1988)), regarding single chain antibodies.

The term “nanobody” (Nb), as used herein, refers to the smallest antigenbinding fragment or single variable domain (V_(HH)) derived fromnaturally occurring heavy chain antibody and is known to the personskilled in the art. They are derived from heavy chain only antibodies,seen in camelids (Hamers-Casterman et al., 1993; Desmyter et al., 1996).In the family of “camelids” immunoglobulins devoid of light polypeptidechains are found. “Camelids” comprise old world camelids (Camelusbactrianus and Camelus dromedarius) and new world camelids (for example,Llama paccos, Llama glama, Llama guanicoe and Llama vicugna). A singlevariable domain heavy chain antibody is referred to herein as a nanobodyor a V_(HH) antibody.

“Antibody fragments” comprise a portion of an intact antibody, forexample, the antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 (1995)); domain antibodies (dAb; Holt et al. (2003)Trends Biotechnol. 21:484); single-chain antibody molecules; andmulti-specific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRS of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The “Fab” fragment also contains the constant domain of the light chainand the first constant domain (CH₁) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH₁ domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains. Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these classes can be further divided intosubclasses (isotypes), e.g., IgGI, IgG2, IgG3, IgG4, IgA, and IgA2. Thesubclasses can be further divided into types, e.g., IgG2a and IgG2b.

“Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise theV_(H) and V_(L) domains of antibody, wherein these domains are presentin a single polypeptide chain. In some embodiments, the Fv polypeptidefurther comprises a polypeptide linker between the V_(H) and V_(L)domains, which enables the sFv to form the desired structure for antigenbinding. For a review of sFv, see Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)—V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

As used herein, the term “affinity” refers to the equilibrium constantfor the reversible binding of two agents (e.g., an antibody and anantigen) and is expressed as a dissociation constant (K_(D)). Affinitycan be at least 1-fold greater, at least 2-fold greater, at least 3-foldgreater, at least 4-fold greater, at least 5-fold greater, at least6-fold greater, at least 7-fold greater, at least 8-fold greater, atleast 9-fold greater, at least 10-fold greater, at least 20-foldgreater, at least 30-fold greater, at least 40-fold greater, at least50-fold greater, at least 60-fold greater, at least 70-fold greater, atleast 80-fold greater, at least 90-fold greater, at least 100-foldgreater, or at least 1,000-fold greater, or more, than the affinity ofan antibody for unrelated amino acid sequences. Affinity of an antibodyto a target protein can be, for example, from about 100 nanomolar (nM)to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or fromabout 100 nM to about 1 femtomolar (fM) or more. As used herein, theterm “avidity” refers to the resistance of a complex of two or moreagents to dissociation after dilution. The terms “immunoreactive” and“preferentially binds” are used interchangeably herein with respect toantibodies and/or antigen-binding fragments.

The term “binding” refers to a direct association between two molecules,due to, for example, covalent, electrostatic, hydrophobic, and ionicand/or hydrogen-bond interactions, including interactions such as saltbridges and water bridges. In some cases, the first member of a specificbinding pair present in the extracellular domain of a chimeric Notchreceptor polypeptide of the present disclosure binds specifically to asecond member of the specific binding pair. “Specific binding” refers tobinding with an affinity of at least about 10⁻⁷ M or greater, e.g.,5×10⁻⁷ M, 10⁻⁸ M, 5×10⁻⁸ M, and greater. “Non-specific binding” refersto binding with an affinity of less than about 10⁻⁷ M, e.g., bindingwith an affinity of 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴ M, etc.

The terms “polypeptide,” “peptide,” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones. The term includes fusionproteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, with or without N-terminal methionineresidues; immunologically tagged proteins; and the like.

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, the polypeptide will bepurified (1) to greater than 90%, greater than 95%, or greater than 98%,by weight of antibody as determined by the Lowry method, for example,more than 99% by weight, (2) to a degree sufficient to obtain at least15 residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (3) to homogeneity by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing ornonreducing conditions using Coomassie blue or silver stain. Isolatedpolypeptide includes the polypeptide in situ within recombinant cellssince at least one component of the polypeptide's natural environmentwill not be present. In some instances, isolated polypeptide will beprepared by at least one purification step.

The terms “chimeric antigen receptor” and “CAR”, used interchangeablyherein, refer to artificial multi-module molecules capable of triggeringor inhibiting the activation of an immune cell which generally but notexclusively comprise an extracellular domain (e.g., a ligand/antigenbinding domain), a transmembrane domain and one or more intracellularsignaling domains. The term CAR is not limited specifically to CARmolecules but also includes CAR variants. CAR variants include splitCARs wherein the extracellular portion (e.g., the ligand bindingportion) and the intracellular portion (e.g., the intracellularsignaling portion) of a CAR are present on two separate molecules. CARvariants also include ON-switch CARs which are conditionally activatableCARs, e.g., comprising a split CAR wherein conditionalhetero-dimerization of the two portions of the split CAR ispharmacologically controlled. CAR variants also include bispecific CARs,which include a secondary CAR binding domain that can either amplify orinhibit the activity of a primary CAR. CAR variants also includeinhibitory chimeric antigen receptors (iCARs) which may, e.g., be usedas a component of a bispecific CAR system, where binding of a secondaryCAR binding domain results in inhibition of primary CAR activation. CARmolecules and derivatives thereof (i.e., CAR variants) are described,e.g., in PCT Application No. US2014/016527; Fedorov et al. Sci TranslMed (2013); 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21;Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al.Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33;Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu RevMed (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98;Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosuresof which are incorporated herein by reference in their entirety.

As used herein, GFP nanobodies may be referred to herein according totheir “LaG” (Llama antibody against GFP) nomenclature according to Fridyet al. (2014) Nat. Methods. 11(12):1253-1260; the disclosure of which,including related supplemental materials, is incorporated herein byreference in its entirety. Accordingly, e.g., in instances where GFP (ormutant of GFP or other Cnidarian fluorescent proteins related to GFP(e.g., AmCFP, DsRed, etc.), is used as an adaptor molecule, variouscombinations of LaG nanobodies may find use provided the members of theLaG nanobody pair do not interfere with one another in their binding toGFP, e.g., where the members of the pair of LaG nanobodies binddifferent epitopes of GFP. LaG nanobodies include but are not limited toe.g., LaG-2, LaG-3, LaG-6, LaG-9, LaG-10, LaG-12, LaG-14, LaG-16,LaG-17, LaG-19, LaG-21, LaG-24, LaG-26, LaG-27, LaG-29, LaG-30, LaG-35,LaG-37, LaG-41, LaG-42, LaG-43, LaG-5, LaG-8, LaG-11, LaG-18,LaG16-G₄S-2, LaG16-3×FLAG-2, LaG41-G₄S-2, and the like. In someinstances, llama antibodies against mCherry (LaM) may also find use inthe systems and devices as described herein where, e.g., LaM nanobodiesinclude but are not limited to e.g., LaM-1, LaM-2, LaM-3, LaM-4, LaM-6,LaM-8.

As used herein, the terms “treatment,” “treating,” “treat” and the like,refer to obtaining a desired pharmacologic and/or physiologic effect.The effect can be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or can be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichcan be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the disease.

The terms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein, refer to a mammal, including, but not limitedto, murines (rats, mice), non-human primates, humans, canines, felines,ungulates (e.g., equines, bovines, ovines, porcines, caprines),lagomorphs, etc. In some cases, the individual is a human. In somecases, the individual is a non-human primate. In some cases, theindividual is a rodent, e.g., a rat or a mouse. In some cases, theindividual is a lagomorph, e.g., a rabbit.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “achimeric Notch receptor polypeptide” includes a plurality of suchchimeric Notch receptor polypeptide and reference to “the geneticallymodified host cell” includes reference to one or more geneticallymodified host cells and equivalents thereof known to those skilled inthe art, and so forth. It is further noted that the claims may bedrafted to exclude any optional element. As such, this statement isintended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides chimeric Notch receptor polypeptides,nucleic acids comprising nucleotide sequences encoding the chimericNotch receptor polypeptides, and host cells genetically modified withthe nucleic acids. A chimeric Notch receptor polypeptide is useful in avariety of applications, which are also provided.

The instant disclosure includes binding-triggered transcriptionalswitches and methods of using binding-triggered transcriptionalswitches. As used herein, a “binding-triggered transcriptional switch”generally refers to a synthetic modular polypeptide or system ofinteracting polypeptides having an extracellular domain that includes afirst member of a specific binding pair, a binding-transducer and anintracellular domain. Upon binding of the second member of the specificbinding pair to the binding-triggered transcriptional switch the bindingsignal is transduced to the intracellular domain such that theintracellular domain becomes activated and performs some function withinthe cell that it does not perform in the absence of the binding signal.

The components of binding-triggered transcriptional switches and thearrangement of the components of the switch relative to one another willvary depending on many factors including but not limited to e.g., thedesired binding trigger, the activity of the intracellular domain, theoverall function of the binding-triggered transcriptional switch, thebroader arrangement of a molecular circuit comprising thebinding-triggered transcriptional switch, etc. The first binding membermay include but is not limited to e.g., those first and/or secondbinding members of specific binding pairs described herein. Theintracellular domain may include but is not limited e.g., thoseintracellular domains and or domains having the biological functions asdescribed herein.

The binding transducer of binding-triggered transcriptional switcheswill also vary depending on the desired method of transduction of thebinding signal. Generally, binding transducers may include thosepolypeptides and/or domains of those polypeptides that transduce anextracellular signal to intracellular signaling e.g., as performed bythe receptors of various signal transduction pathways. Transduction of abinding signal may be achieved through various mechanisms including butnot limited to e.g., binding-induced proteolytic cleavage,binding-induced phosphorylation, binding-induced conformational change,etc. In some instances, a binding-transducer may contain aligand-inducible proteolytic cleavage site such that upon binding thebinding-signal is transduced by cleavage of the binding-triggeredtranscriptional switch, e.g., to liberate an intracellular domain. Forexample, in some instances, a binding-triggered transcriptional switchmay include a Notch derived cleavable binding transducer, such as, e.g.,a chimeric notch receptor polypeptide as described herein.

In other instances, the binding signal may be transduced in the absenceof inducible proteolytic cleavage. Any signal transduction component orcomponents of a signaling transduction pathway may find use in abinding-triggered transcriptional switch whether or not proteolyticcleavage is necessary for signal propagation. For example, in someinstances, a phosphorylation-based binding transducer, including but notlimited to e.g., one or more signal transduction components of theJak-Stat pathway, may find use in a non-proteolytic binding-triggeredtranscriptional switch.

For simplicity, binding-triggered transcriptional switches, includingbut not limited to chimeric notch receptor polypeptides, are describedprimarily as single polypeptide chains. However, as will be clear fromthe instant disclosure, binding-triggered transcriptional switches,including chimeric notch receptor polypeptides, may be divided or splitacross two or more separate polypeptide chains where the joining of thetwo or more polypeptide chains to form a functional binding-triggeredtranscriptional switch, e.g., a chimeric notch receptor polypeptide, maybe constitutive or conditionally controlled. For example, constitutivejoining of two portions of a split binding-triggered transcriptionalswitch may be achieved by inserting a constitutive heterodimerizationdomain between the first and second portions of the split polypeptidesuch that upon heterodimerization the split portions are functionallyjoined.

In some instances, the joining of a split binding-triggeredtranscriptional switch and/or the signaling from a splitbinding-triggered transcriptional switch may be conditionally controlledthrough the use of an “adapter” that mediates the functional joining,e.g., of first and second parts of a split binding-triggeredtranscriptional switch. To mediate signaling through a splitbinding-triggered transcriptional switch the adapter may be added oradministered directly or may be indirectly produced, e.g., throughexpression of the adapter from a cell configured for such expression,e.g., either conditionally or constitutively. Useful adapters includethose proteins having a first and second binding surface that can besimultaneously utilized by two different binding molecules. In someinstances, adapters may include proteins for which two antibodies bindto two different epitopes of the protein.

For example, in some instances, an antigen may find use as an adaptorwhere the binding molecules utilized may be two different antibodiesthat bind to two different epitopes of the antigen. In such aconfiguration, attachment of the antibodies, or portions thereof, to thefirst and second parts of the split binding-triggered transcriptionalswitch results in functional joining of the parts in the presence of theantigen as mediated by simultaneous binding of both antibodies to asingle molecule of the antigen. Antigens that can function as adaptorsinclude, but are not limited to, antigens of a pathogen,cancer-associated antigens, disease-associated antigens, antibodies, andthe like. In some cases, the adaptor antigen is soluble (e.g., not boundto the surface of a cell). In some cases, the adaptor antigen is boundto the surface of a cell.

For example, in some instances GFP may find use as an adaptor where thebinding molecules utilized may be two different antibodies that bind totwo different surfaces of GFP. In such a configuration, attachment ofthe antibodies, or portions thereof, to the first and second parts ofthe split binding-triggered transcriptional switch results in functionaljoining of the parts in the presence of GFP as mediated by simultaneousbinding of both antibodies to a single molecule of GFP.

In some instances, a split binding-triggered transcriptional switch,e.g., where binding of the first and second parts of the splitbinding-triggered transcriptional switch is mediated by an antigenadaptor and results in functional joining of the parts, the splitbinding-triggered transcriptional switch allows for detection of thepresence of the antigen in the vicinity of the first and second parts ofthe split binding-triggered transcriptional switch. For example, incertain embodiments, a first part of a binding-triggered transcriptionalswitch expressed on the surface of a first cell and a second part of abinding-triggered transcriptional switch is expressed on a second celland such first and second parts are configured such that when a solubleantigen is present in the vicinity of the first and second cells thefirst and second parts are functionally joined by the antigen resultingin activation of a reporter by the activated binding-triggeredtranscriptional switch.

Both parts of the split binding-triggered transcriptional switch neednot necessarily be anchored to a cell to function in the detection of anantigen. For example, in some instances, a first part of abinding-triggered transcriptional switch is solubly expressed and asecond part of a binding-triggered transcriptional switch is expressedon a cell and such first and second parts are configured such that whena soluble antigen is present in the vicinity of the first and secondparts the parts are functionally joined by the antigen resulting inpriming of the binding-triggered transcription switch making the primedbinding-triggered transcription switch capable of responding, e.g.,reporting, a second event including e.g., the presence of a secondantigen that activates the binding-triggered transcriptional switch.Such a second antigen may be present on the surface of a cell or may notbe attached to a cell (i.e., soluble).

Conditional control of the joining of the portions of a splitbinding-triggered transcriptional switch provides further control ofsignaling from the split binding-triggered transcriptional switch. Forexample, by mediating joining by providing or expressing an adaptorsignaling from the split binding-triggered transcriptional switch asituation permissive to signaling from the switch is created.Conversely, by inhibiting joining by providing a competitive inhibitorthat prevents joining of the portions of a split binding-triggeredtranscriptional switch a signaling from the switch may be prevented. Insome instances, such effects are dose dependent, i.e., can be furthercontrolled based on the amount of provided adapter and/or competitiveinhibitor.

Accordingly, given the descriptions of split binding-triggeredtranscriptional switches provided herein and the descriptions of singlepolypeptide binding-triggered transcriptional switches an ordinaryskilled artisan will readily understand wherein split polypeptides maybe utilized to provide additional constitutive and/or conditionalcontrol over the signaling from such switches and molecular circuitscontaining such switches.

Chimeric Notch Receptor Polypeptides

The present disclosure provides chimeric Notch receptor polypeptides. Achimeric Notch receptor polypeptide of the present disclosure comprises:a) an extracellular domain comprising a first member of a specificbinding pair; b) a Notch receptor polypeptide, where the Notch receptorpolypeptide has a length of from 50 amino acids to 1000 amino acids, andcomprises one or more ligand-inducible proteolytic cleavage sites; andc) an intracellular domain. Binding of the first member of the specificbinding pair to a second member of the specific binding pair inducescleavage of the Notch receptor polypeptide at the one or moreligand-inducible proteolytic cleavage sites, thereby releasing theintracellular domain. Release of the intracellular domain modulates anactivity of a cell that produces the chimeric Notch receptorpolypeptide. The extracellular domain comprises a first member of aspecific binding pair; the first member of a specific binding paircomprises an amino acid sequence that is heterologous to the Notchreceptor polypeptide. The intracellular domain comprises an amino acidsequence that is heterologous to the Notch receptor polypeptide.

A schematic depiction of a Notch receptor polypeptide is provided inFIG. 1. The Notch receptor polypeptide depicted in FIG. 1 includes: a)an extracellular portion that includes: i) epidermal growth factor (EGF)repeats; ii) a ligand binding site; iii) three Lin-12 Notch repeats(LNR), designated LNR-A, LNR-B, and LNR-C; iv) two heterodimerizationdomains (HD-N and HD-C); b) a transmembrane (TM) portion; and c) anintracellular portion that includes: i) a RAM domain; ii) ankyrinrepeats; iii) a transcription activation domain; and iv) a PEST region.A Notch receptor polypeptide includes three proteolytic sites, termedS1, S2, and S3. S1, a furin cleavage site, is located between HD-N andHC-C; S2, an ADAM17 cleavage site, is located within HD-C; and S3, agamma secretase cleavage site, is within the TM portion. A Notchreceptor polypeptide mediates cell-to-cell communication, e.g.communication between contacting cells, in which one contacting cell isa “receiver” cell and the other contacting cell is a “sender” cell.Engagement of a Notch receptor polypeptide present on a receiving cellby a Delta polypeptide (“ligand”) present on a sending cell results inligand-induced cleavage of the Notch receptor polypeptide, resulting inrelease of the intracellular portion of the receptor from the membraneinto the cytoplasm. The released portion alters receiver cell behaviorby functioning as a transcriptional regulator.

Extracellular Domain

As noted above, a chimeric Notch receptor polypeptide of the presentdisclosure comprises an extracellular domain. The extracellular domaincomprises a first member of a specific binding pair. The first member ofthe specific binding pair binds to a second member of the specificbinding pair, where the second member of the specific binding pair is ona polypeptide that is different from the chimeric Notch receptorpolypeptide of the present disclosure. The second member of the specificbinding pair is separate from (e.g., not covalently linked to) thechimeric Notch receptor polypeptide comprising extracellular domaincomprises a first member of the specific binding pair. The second memberof the specific binding pair can be present on the surface of a cell.The second member of the specific binding pair can be immobilized on aninsoluble support. The second member of the specific binding pair can besoluble. The second member of the specific binding pair can be presentin an extracellular environment (e.g., extracellular matrix). The secondmember of the specific binding pair can be present in an artificialmatrix. The second member of the specific binding pair can be present inan acellular environment.

The extracellular domain comprises a first member of a specific bindingpair that is heterologous to the Notch receptor polypeptide. In otherwords, the first member of the specific binding pair present in theextracellular domain is not naturally present in a Notch receptorpolypeptide.

Suitable first members of a specific binding pairs include, but are notlimited to, antibody-based recognition scaffolds; antibodies (i.e., anantibody-based recognition scaffold, including antigen-binding antibodyfragments); non-antibody-based recognition scaffolds; antigens (e.g.,endogenous antigens; exogenous antigens; etc.); a ligand for a receptor;a receptor; a target of a non-antibody-based recognition scaffold; an Fcreceptor (e.g., FcγRIIIa; FcγRIIIb; etc.); an extracellular matrixcomponent; and the like.

Specific binding pairs include, e.g., antigen-antibody specific bindingpairs, where the first member is an antibody (or antibody-basedrecognition scaffold) that binds specifically to the second member,which is an antigen, or where the first member is an antigen and thesecond member is an antibody (or antibody-based recognition scaffold)that binds specifically to the antigen; ligand-receptor specific bindingpairs, where the first member is a ligand and the second member is areceptor to which the ligand binds, or where the first member is areceptor, and the second member is a ligand that binds to the receptor;non-antibody-based recognition scaffold-target specific binding pairs,where the first member is a non-antibody-based recognition scaffold andthe second member is a target that binds to the non-antibody-basedrecognition scaffold, or where the first member is a target and thesecond member is a non-antibody-based recognition scaffold that binds tothe target; adhesion molecule-extracellular matrix binding pairs; Fcreceptor-Fc binding pairs, where the first member comprises animmunoglobulin Fc that binds to the second member, which is an Fcreceptor, or where the first member is an Fc receptor that binds to thesecond member which comprises an immunoglobulin Fc; andreceptor-co-receptor binding pairs, where the first member is a receptorthat binds specifically to the second member which is a co-receptor, orwhere the first member is a co-receptor that binds specifically to thesecond member which is a receptor.

Non-limiting examples of suitable extracellular domains include, e.g.,Cadherins (CDH1-20), Integrins (alfa and beta isoforms), Ephrins, NCAMs,connexins, CD44, syndecan, CD47, DGalfa/beta, SV2, protocadherin, Fas,Dectin-1, CD7, CD40, Neuregulin, KIR, BTLA, Tim-2, Lag-3, CD19, CTLA4,CD28, TIGIT, and ICOS.

In some cases, the extracellular domain comprises a toll-like receptor(TLR). In some cases, the extracellular domain comprises a dectin thatrecognizes N-glycans that are present on the surface of pathogenic fungiand cancer cells. See, e.g., Xie (2012) Glycoconj. 29:273; and Brown etal. (2007) Protein Sci. 16:1042. In some cases, the extracellular domaincomprises a polypeptide that recognizes a bacterial surface molecule.

In some cases, the extracellular domain of a chimeric Notch polypeptideof the present disclosure comprises an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to the .. . following amino acid sequence:

(SEQ ID NO: 3) GVLSSPCPPNWIIYEKSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDSSNELGFIVKQVSSQPDNSFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIYDQLCSVPSYSICEKKFSM.

A skilled artisan can select an extracellular domain based on thedesired localization or function of a cell that is genetically modifiedto express a chimeric Notch receptor polypeptide of the presentdisclosure. For example, the extracellular domain can target cells toestrogen-dependent breast cancer cells that have an increased number ofestrogen receptors on the cell surface, where the first member of thespecific binding pair binds to an estrogen receptor (second member ofthe specific binding pair). Other non-limiting examples ofligand/receptor interactions include CCRI (e.g., for targeting toinflamed joint tissues or brain in rheumatoid arthritis, and/or multiplesclerosis), CCR7, CCR8 (e.g., targeting to lymph node tissue), CCR6,CCR9, CCRIO (e.g., to target to intestinal tissue), CCR4, CCRIO (e.g.,for targeting to skin), CXCR4 (e.g., for general enhancedtransmigration), HCELL (e.g., for targeting of inflammation andinflammatory disorders, bone marrow), Alpha4beta7 (e.g., for intestinalmucosa targeting), VLA-4/VCAM-I (e.g., targeting to endothelium). Ingeneral, any receptor involved in targeting (e.g., cancer metastasis)can be used as an extracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure.

Antibody-Based Recognition Scaffolds

In some cases, the first member of the specific binding pair is anantibody. The antibody can be any antigen-binding antibody-basedpolypeptide, a wide variety of which are known in the art. In someinstances, the antigen-binding domain is a single chain Fv (scFv). Otherantibody based recognition domains (cAb VHH (camelid antibody variabledomains) and humanized versions, IgNAR VH (shark antibody variabledomains) and humanized versions, sdAb VH (single domain antibodyvariable domains) and “camelized” antibody variable domains are suitablefor use. In some instances, T-cell receptor (TCR) based recognitiondomains such as single chain TCR (scTv, single chain two-domain TCRcontaining VaV) are also suitable for use.

Where the member of a specific binding pair in a chimeric Notch receptorpolypeptide of the present disclosure is an antibody-based recognitionscaffold, the chimeric Notch receptor polypeptide can be activated inthe presence of a second member of the specific binding pair, where thesecond member of the specific binding pair is an antigen that binds tothe antibody-based recognition scaffold.

An antibody suitable for inclusion in a chimeric Notch polypeptide ofthe present disclosure can have a variety of antigen-bindingspecificities.

In some cases, the antigen-binding domain is specific for an epitopepresent in an antigen that is expressed by (synthesized by) a cancercell, i.e., a cancer cell associated antigen. The cancer cell associatedantigen can be an antigen associated with, e.g., a breast cancer cell, aB cell lymphoma, a pancreatic cancer, a Hodgkin lymphoma cell, anovarian cancer cell, a prostate cancer cell, a mesothelioma, a lungcancer cell (e.g., a small cell lung cancer cell), a non-Hodgkin B-celllymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, amesothelioma cell, a lung cancer cell (e.g., a small cell lung cancercell), a melanoma cell, a chronic lymphocytic leukemia cell, an acutelymphocytic leukemia cell, a neuroblastoma cell, a glioma, aglioblastoma, a medulloblastoma, a colorectal cancer cell, etc. A cancercell associated antigen may also be expressed by a non-cancerous cell.

In some cases, the antigen-binding domain is specific for an epitopepresent in a tissue-specific antigen. In some cases, the antigen-bindingdomain is specific for an epitope present in a disease-associatedantigen.

Non-limiting examples of antigens to which an antigen-binding domain ofa subject chimeric Notch receptor polypeptide can bind include, e.g.,CD19, CD20, CD38, CD30, Her2/neu, ERBB2, CA125, MUC-1, prostate-specificmembrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin,carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR),EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), highmolecular weight-melanoma associated antigen (HMW-MAA), MAGE-A1,IL-13R-a2, GD2, and the like.

Non-limiting examples of antigens to which an antigen-binding domain ofa subject chimeric Notch receptor polypeptide can bind include, e.g.,Cadherins (CDH1-20), Integrins (alfa and beta isoforms), Ephrins, NCAMs,connexins, CD44, syndecan, CD47, DGalfa/beta, SV2, protocadherin, Fas,Dectin-1, CD7, CD40, Neuregulin, KIR, BTLA, Tim-2, Lag-3, CD19, CTLA4,CD28, TIGIT, and ICOS.

In some cases, the antibody is specific for a cytokine. In some cases,the antibody is specific for a cytokine receptor. In some cases, theantibody is specific for a growth factor. In some cases, the antibody isspecific for a growth factor receptor. In some cases, the antibody isspecific for a cell-surface receptor.

In some cases, the antibody is specific for a cell surface target, wherenon-limiting examples of cell surface targets include CD19, CD30, Her2,CD22, ENPP3, EGFR, CD20, CD52, CD 11a, and alpha-integrin.

In some cases, the antigen (second member of the specific binding pair)bound by the antibody-based scaffold is soluble. In some cases, theantigen is membrane-bound, e.g., in some cases, the antigen is presenton the surface of a cell. In some cases, the antigen is immobilized onan insoluble support, where an insoluble support can comprise any of avariety of materials (e.g., polyethylene, polystyrene,polyvinylpyrrolidone, polycarbonate, nitrocellulose, and the like); andwhere an insoluble support can take a variety of forms, e.g., a plate, atissue culture dish, a column, and the like. In some cases, the antigenis present in an extracellular matrix (ECM) (e.g., the antigen is an ECMcomponent). In some cases, the antigen is present in an artificialmatrix. In some cases, the antigen is present in an acellularenvironment.

Non-Antibody-Based Recognition Scaffolds

In some cases, the first member of the specific binding pair is anon-antibody-based recognition scaffold. Where the member of a specificbinding pair in a chimeric Notch receptor polypeptide of the presentdisclosure is a non-antibody-based recognition scaffold, the chimericNotch receptor polypeptide can be activated in the presence of a secondmember of the specific binding pair, where the second member of thespecific binding pair is a target that binds to the non-antibody-basedrecognition scaffold.

Non-antibody-based recognition scaffolds include, e.g., an affibodies;engineered Kunitz domains; monobodies (adnectins); anticalins; designedankyrin repeat domains (DARPins); a binding site of a cysteine-richpolypeptide (e.g., cysteine-rich knottin peptides); avimers; afflins;and the like. See, e.g., Gebauer and Skerra (2009) Curr. Opin. Chem.Biol. 13:245.

Non-antibody-based scaffolds (also referred to herein as “antibody mimicmolecules”) may be identified by selection or isolation of atarget-binding variant from a library of binding molecules havingartificially diversified binding sites. Diversified libraries can begenerated using completely random approaches (e.g., error-pronepolymerase chain reaction (PCR), exon shuffling, or directed evolution)or aided by art-recognized design strategies. For example, amino acidpositions that are usually involved when the binding site interacts withits cognate target molecule can be randomized by insertion of degeneratecodons, trinucleotides, random peptides, or entire loops atcorresponding positions within the nucleic acid which encodes thebinding site (see e.g., U.S. Pub. No. 20040132028). The location of theamino acid positions can be identified by investigation of the crystalstructure of the binding site in protein entity with the targetmolecule. Candidate positions for randomization include loops, flatsurfaces, helices, and binding cavities of the binding site. In certainembodiments, amino acids within the binding site that are likelycandidates for diversification can be identified by their homology withthe immunoglobulin fold. For example, residues within the CDR-like loopsof fibronectin may be randomized to generate a library of fibronectinbinding molecules (see, e.g., Koide et al., J. Mol. Biol., 284:1141-1151 (1998)). Other portions of the binding site which may berandomized include flat surfaces. Following randomization, thediversified library may then be subjected to a selection or screeningprocedure to obtain binding molecules with the desired bindingcharacteristics. For example, selection can be achieved byart-recognized methods such as phage display, yeast display, or ribosomedisplay.

For example, in some cases, the non-antibody-based scaffold comprises abinding site from a fibronectin binding molecule. Fibronectin bindingmolecules (e.g., molecules comprising the Fibronectin type I, II, or IIIdomains) display CDR-like loops which, in contrast to immunoglobulins,do not rely on intra-chain disulfide bonds. The FnIII loops compriseregions that may be subjected to random mutation and directedevolutionary schemes of iterative rounds of target binding, selection,and further mutation in order to develop useful therapeutic tools.Fibronectin-based “addressable” therapeutic binding molecules (“FATBIM”)can be developed to specifically bind the target antigen or epitope.Methods for making fibronectin binding polypeptides are described, forexample, in WO 01/64942 and in U.S. Pat. Nos. 6,673,901, 6,703,199,7,078,490, and 7,119,171.

As another example, in some cases, the non-antibody-based scaffoldcomprises a binding site from an affibody. Affibodies are derived fromthe immunoglobulin binding domains of staphylococcal Protein A (SPA)(see e.g., Nord et al., Nat. Biotechnol., 15: 772-777 (1997)). Anaffibody is an antibody mimic that has unique binding sites that bindspecific targets. Affibodies can be small (e.g., consisting of threealpha helices with 58 amino acids and having a molar mass of about 6kDa), have an inert format (no Fc function), and have been successfullytested in humans as targeting moieties. Affibody binding sites can besynthesized by mutagenizing an SPA-related protein (e.g., Protein Z)derived from a domain of SPA (e.g., domain B) and selecting for mutantSPA-related polypeptides having binding affinity for a target antigen orepitope. Other methods for making affibody binding sites are describedin U.S. Pat. Nos. 6,740,734 and 6,602,977 and in WO 00/63243.

As another example, in some cases, the non-antibody-based scaffoldcomprises a binding site from an anticalin. An anticalin is an antibodyfunctional mimetic derived from a human lipocalin. Lipocalins are afamily of naturally-occurring binding proteins that bind and transportsmall hydrophobic molecules such as steroids, bilins, retinoids, andlipids. The main structure of an anticalin is similar to wild typelipocalins. The central element of this protein architecture is abeta-barrel structure of eight antiparallel strands, which supports fourloops at its open end. These loops form the natural binding site of thelipocalins and can be reshaped in vitro by extensive amino acidreplacement, thus creating novel binding specificities. Anticalinspossess high affinity and specificity for their ligands as well as fastbinding kinetics, so that their functional properties are similar tothose of antibodies. Anticalins are described in, e.g., U.S. Pat. No.7,723,476.

As another example, in some cases, the non-antibody-based scaffoldcomprises a binding site from a cysteine-rich polypeptide. Cysteine-richdomains in some cases do not form an alpha-helix, a beta-sheet, or abeta-barrel structure. In some cases, the disulfide bonds promotefolding of the domain into a three-dimensional structure. In some cases,cysteine-rich domains have at least two disulfide bonds, e.g., at leastthree disulfide bonds. An exemplary cysteine-rich polypeptide is an Adomain protein. A-domains (sometimes called “complement-type repeats”)contain about 30-50 or 30-65 amino acids. In some cases, the domainscomprise about 35-45 amino acids and in some cases about 40 amino acids.Within the 30-50 amino acids, there are about 6 cysteine residues. Ofthe six cysteines, disulfide bonds typically are found between thefollowing cysteines: Cl and C3, C2 and C5, C4 and C6. The A domainconstitutes a ligand binding moiety. The cysteine residues of the domainare disulfide linked to form a compact, stable, functionally independentmoiety. Clusters of these repeats make up a ligand binding domain, anddifferential clustering can impart specificity with respect to theligand binding. Exemplary proteins containing A-domains include, e.g.,complement components (e.g., C6, C7, C8, C9, and Factor I), serineproteases (e.g., enteropeptidase, matriptase, and corin), transmembraneproteins (e.g., ST7, LRP3, LRP5 and LRP6) and endocytic receptors (e.g.Sortilin-related receptor, LDL-receptor, VLDLR, LRP1, LRP2, and ApoER2).Methods for making A-domain proteins of a desired binding specificityare disclosed, for example, in WO 02/088171 and WO 04/044011.

As another example, in some cases, the non-antibody-based scaffoldcomprises a binding site from a repeat protein. Repeat proteins areproteins that contain consecutive copies of small (e.g., about 20 toabout 40 amino acid residues) structural units or repeats that stacktogether to form contiguous domains. Repeat proteins can be modified tosuit a particular target binding site by adjusting the number of repeatsin the protein. Exemplary repeat proteins include designed ankyrinrepeat proteins (i.e., a DARPins) (see e.g., Binz et al., Nat.Biotechnol., 22: 575-582 (2004)) or leucine-rich repeat proteins (i.e.,LRRPs) (see e.g., Pancer et al., Nature, 430: 174-180 (2004)). Asanother example, in some cases, the non-antibody-based scaffoldcomprises a DARPin.

As used herein, the term “DARPin” refers to a genetically engineeredantibody mimetic protein that typically exhibits highly specific andhigh-affinity target protein binding. DARPins were first derived fromnatural ankyrin proteins. In some cases, DARPins comprise three, four orfive repeat motifs of an ankyrin protein. In some cases, a unit of anankyrin repeat consists of 30-34 amino acid residues and functions tomediate protein-protein interactions. In some cases, each ankyrin repeatexhibits a helix-turn-helix conformation, and strings of such tandemrepeats are packed in a nearly linear array to form helix-turn-helixbundles connected by relatively flexible loops. In some cases, theglobal structure of an ankyrin repeat protein is stabilized by intra-and inter-repeat hydrophobic and hydrogen bonding interactions. Therepetitive and elongated nature of the ankyrin repeats provides themolecular bases for the unique characteristics of ankyrin repeatproteins in protein stability, folding and unfolding, and bindingspecificity. The molecular mass of a DARPin domain can be from about 14or 18 kDa for four- or five-repeat DARPins, respectively. DARPins aredescribed in, e.g., U.S. Pat. No. 7,417,130. In some cases, tertiarystructures of ankyrin repeat units share a characteristic composed of abeta-hairpin followed by two antiparallel alpha-helices and ending witha loop connecting the repeat unit with the next one. Domains built ofankyrin repeat units can be formed by stacking the repeat units to anextended and curved structure. LRRP binding sites from part of theadaptive immune system of sea lampreys and other jawless fishes andresemble antibodies in that they are formed by recombination of a suiteof leucine-rich repeat genes during lymphocyte maturation. Methods formaking DARpin or LRRP binding sites are described in WO 02/20565 and WO06/083275.

As another example, in some cases, the non-antibody-based scaffoldcomprises a binding site derived from Src homology domains (e.g. SH2 orSH3 domains), PDZ domains, beta-lactamase, high affinity proteaseinhibitors, or small disulfide binding protein scaffolds such asscorpion toxins. Methods for making binding sites derived from thesemolecules have been disclosed in the art, see e.g., Panni et al., J.Biol. Chem., 277: 21666-21674 (2002), Schneider et al., Nat.Biotechnol., 17: 170-175 (1999); Legendre et al., Protein Sci.,11:1506-1518 (2002); Stoop et al., Nat. Biotechnol., 21: 1063-1068(2003); and Vita et al., PNAS, 92: 6404-6408 (1995). Yet other bindingsites may be derived from a binding domain selected from the groupconsisting of an EGF-like domain, a Kringle-domain, a PAN domain, a Gladomain, a SRCR domain, a Kunitz/Bovine pancreatic trypsin Inhibitordomain, a Kazal-type serine protease inhibitor domain, a Trefoil(P-type) domain, a von Willebrand factor type C domain, anAnaphylatoxin-like domain, a CUB domain, a thyroglobulin type I repeat,LDL-receptor class A domain, a Sushi domain, a Link domain, aThrombospondin type I domain, an Immunoglobulin-like domain, a C-typelectin domain, a MAM domain, a von Willebrand factor type A domain, aSomatomedin B domain, a WAP-type four disulfide core domain, a F5/8 typeC domain, a Hemopexin domain, a Laminin-type EGF-like domain, a C2domain, a binding domain derived from tetranectin in its monomeric ortrimeric form, and other such domains known to those of ordinary skillin the art, as well as derivatives and/or variants thereof. Exemplarynon-antibody-based scaffolds, and methods of making the same, can alsobe found in Stemmer et al., “Protein scaffolds and uses thereof”, U.S.Patent Publication No. 20060234299 (Oct. 19, 2006) and Hey, et al.,Artificial, Non-Antibody Binding Proteins for Pharmaceutical andIndustrial Applications, TRENDS in Biotechnology, vol. 23, No. 10, Table2 and pp. 514-522 (October 2005).

As another example, in some cases, the non-antibody-based scaffoldcomprises a Kunitz domain. The term “Kunitz domains” as used herein,refers to conserved protein domains that inhibit certain proteases,e.g., serine proteases. Kunitz domains are relatively small, typicallybeing about 50 to 60 amino acids long and having a molecular weight ofabout 6 kDa. Kunitz domains typically carry a basic charge and arecharacterized by the placement of two, four, six or eight or more thatform disulfide linkages that contribute to the compact and stable natureof the folded peptide. For example, many Kunitz domains have sixconserved cysteine residues that form three disulfide linkages. Thedisulfide-rich a/P fold of a Kunitz domain can include two, three(typically), or four or more disulfide bonds.

Kunitz domains have a pear-shaped structure that is stabilized the,e.g., three disulfide bonds, and that contains a reactive site regionfeaturing the principal determinant P1 residue in a rigid confirmation.These inhibitors competitively prevent access of a target protein (e.g.,a serine protease) for its physiologically relevant macromolecularsubstrate through insertion of the P1 residue into the active sitecleft. The P1 residue in the proteinase-inhibitory loop provides theprimary specificity determinant and dictates much of the inhibitoryactivity that particular Kunitz protein has toward a targetedproteinase. In general, the N-terminal side of the reactive site (P) isenergetically more important that the PC-terminal side. In most cases,lysine or arginine occupy the P1 position to inhibit proteinases thatcleave adjacent to those residues in the protein substrate. Otherresidues, particularly in the inhibitor loop region, contribute to thestrength of binding. Generally, about 10-12 amino acid residues in thetarget protein and 20-25 residues in the proteinase are in directcontact in the formation of a stable proteinase-inhibitor protein entityand provide a buried area of about 600 to 900 A. By modifying theresidues in the P site and surrounding residues Kunitz domains can bedesigned to target a protein of choice. Kunitz domains are described in,e.g., U.S. Pat. No. 6,057,287.

As another example, in some cases, the non-antibody-based scaffold is anaffilin Affilins are small antibody-mimic proteins which are designedfor specific affinities towards proteins and small compounds. Newaffilins can be very quickly selected from two libraries, each of whichis based on a different human derived scaffold protein. Affilins do notshow any structural homology to immunoglobulin proteins. There are twocommonly-used affilin scaffolds, one of which is gamma crystalline, ahuman structural eye lens protein and the other is “ubiquitin”superfamily proteins. Both human scaffolds are very small, show hightemperature stability and are almost resistant to pH changes anddenaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in WO200104144 and examples of“ubiquitin-like” proteins are described in WO2004106368.

As another example, in some cases, the non-antibody-based scaffold is anAvimer. Avimers are evolved from a large family of human extracellularreceptor domains by in vitro exon shuffling and phage display,generating multidomain proteins with binding and inhibitory properties.Linking multiple independent binding domains has been shown to createavidity and results in improved affinity and specificity compared withconventional single-epitope binding proteins. In certain embodiments,Avimers consist of two or more peptide sequences of 30 to 35 amino acidseach, connected by spacer region peptides. The individual sequences arederived from A domains of various membrane receptors and have a rigidstructure, stabilized by disulfide bonds and calcium. Each A domain canbind to a certain epitope of the target protein. The combination ofdomains binding to different epitopes of the same protein increasesaffinity to this protein, an effect known as avidity (hence the name).Avimers with sub-nanomolar affinities have been obtained against avariety of targets. Alternatively, the domains can be directed againstepitopes on different target proteins. Additional information regardingavimers can be found in U.S. patent application Publication Nos.2006/0286603, 2006/0234299, 2006/0223114, 2006/0177831, 2006/0008844,2005/0221384, 2005/0164301, 2005/0089932, 2005/0053973, 2005/0048512,2004/0175756.

Suitable targets of a non-antibody-based scaffold include any of theabove-mentioned antigens to which an antibody-based scaffold can bind.

In some cases, the target (second member of the specific binding pair)bound by the non-antibody-based scaffold is soluble. In some cases, thetarget is membrane-bound, e.g., in some cases, the target is present onthe surface of a cell. In some cases, the target is immobilized on aninsoluble support, where an insoluble support can comprise any of avariety of materials (e.g., polyethylene, polystyrene,polyvinylpyrrolidone, polycarbonate, nitrocellulose, and the like); andwhere an insoluble support can take a variety of forms, e.g., a plate, atissue culture dish, a column, and the like. In some cases, the targetis present in an extracellular matrix (ECM) (e.g., the antigen is an ECMcomponent). In some cases, the target is present in an artificialmatrix. In some cases, the target is present in an acellularenvironment.

Cell Adhesion Molecules

In some cases, the first member of the specific binding pair is a celladhesion molecule (CAM), i.e., a polypeptide that binds a component ofan extracellular matrix (ECM) or that binds a cell surface molecule. Forexample, in some cases, the first member of the specific binding pair isthe extracellular region of a CAM. In some cases, the CAM is acalcium-independent adhesion molecule; for example, in some cases, theCAM is an immunoglobulin superfamily CAM. In some cases, the CAM is acalcium-dependent adhesion molecule; e.g., the CAM is an integrin, acadherin, or a selectin. In some cases, the first member of the specificbinding pair is an integrin. In some cases, the first member of thespecific binding pair is a cadherin, e.g., an E-cadherin, a P-cadherin,an N-cadherin, an R-cadherin, an M-cadherin, etc. In some cases, thefirst member of the specific binding pair is a selectin, e.g., anE-selectin, an L-selectin, or a P-selectin. Binding fragments of a CAMcan be used as the first member of the specific binding pair.

Where the first member of the specific binding pair is a CAM, the secondmember of the specific binding pair is a component of ECM or a cellsurface molecule that binds the CAM. For example, where the first memberof the specific binding pair is an integrin, the second member of thespecific binding pair is a component of collagen, fibrinogen,fibronectin, or vitronectin. As another example, where the first memberof the specific binding pair is cadherin, the second member of thespecific binding pair is cell surface antigen bound by the cadherin. Asanother example, where the first member of the specific binding pair isa selectin, the second member of the specific binding pair is afucosylated carbohydrate.

Ligands

In some cases, the first member of the specific binding pair is a ligandfor a receptor. Ligands include polypeptides, nucleic acids,glycoproteins, small molecules, carbohydrates, lipids, glycolipids,lipoproteins, lipopolysaccharides, etc. In some cases, the ligand issoluble.

Ligands include, but are not limited to, cytokines (e.g., IL-13, etc.);growth factors (e.g., heregulin; vascular endothelial growth factor(VEGF); and the like); peptide hormones; an integrin-binding peptide(e.g., a peptide comprising the sequence Arg-Gly-Asp); an N-glycan; andthe like.

Where the member of a specific binding pair in a chimeric Notch receptorpolypeptide of the present disclosure is a ligand, the chimeric Notchreceptor polypeptide can be activated in the presence of a second memberof the specific binding pair, where the second member of the specificbinding pair is a receptor for the ligand. For example, where the ligandis VEGF, the second member of the specific binding pair can be a VEGFreceptor, including a soluble VEGF receptor. Alternatively, the firstmember of the specific binding pair can be a VEGF receptor; and thefirst member of the specific binding pair can be VEGF. As anotherexample, where the ligand is heregulin, the second member of thespecific binding pair can be Her2.

Where the first member of the specific binding pair is a ligand, thesecond member of the specific binding pair is a molecule that binds theligand, e.g., the second member of the specific binding pair is anantibody that specifically binds the ligand, a receptor for the ligand,etc.

Where the first member of the specific binding pair is a ligand, in somecases, the second member of the specific binding pair (the molecule thatbinds the ligand) is soluble. In some cases, the second member of thespecific binding pair is membrane-bound, e.g., in some cases, the secondmember of the specific binding pair is present on the surface of a cell.In some cases, the second member of the specific binding pair isimmobilized on an insoluble support, where an insoluble support cancomprise any of a variety of materials (e.g., polyethylene, polystyrene,polyvinylpyrrolidone, polycarbonate, nitrocellulose, and the like); andwhere an insoluble support can take a variety of forms, e.g., a plate, atissue culture dish, a column, and the like. In some cases, the secondmember of the specific binding pair is present in an acellularenvironment.

Antigens

In some cases, the first member of the specific binding pair is anantigen to which an antibody specifically binds. The antigen can be anyantigen, e.g., a naturally-occurring (endogenous) antigen; a synthetic(e.g., modified in such a way that it is no longer the same as anaturally-occurring antigen; modified from its natural state; etc.)antigen; etc.

Where the member of a specific binding pair in a chimeric Notch receptorpolypeptide of the present disclosure is an antigen, the chimeric Notchreceptor polypeptide can be activated in the presence of a second memberof the specific binding pair, where the second member of the specificbinding pair is an antibody (antibody-based recognition scaffold) thatbinds to the antigen.

In some cases, the antigen is a disease-associated antigen, e.g., acancer-associated antigen, an autoimmune disease-associated antigen, apathogen-associated antigen, an inflammation-associated antigen, or thelike.

For example, where the second member of the specific binding pair is anantibody specific for a cancer-associated antigen, the antigen can be acancer-associated antigen, where cancer-associated antigens include,e.g., CD19, CD20, CD38, CD30, Her2/neu, ERBB2, CA125, MUC-1,prostate-specific membrane antigen (PSMA), CD44 surface adhesionmolecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growthfactor receptor (EGFR), EGFRvIII, vascular endothelial growth factorreceptor-2 (VEGFR2), high molecular weight-melanoma associated antigen(HMW-MAA), MAGE-A1, IL-13R-a2, GD2, and the like. Cancer-associatedantigens also include, e.g., 4-1BB, 5T4, adenocarcinoma antigen,alpha-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonicanhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221,CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6,CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DRS, EGFR, EpCAM,CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factorreceptor kinase, IGF-1 receptor, IGF-I, IgGI, L1-CAM, IL-13, IL-6,insulin-like growth factor I receptor, integrin α5β1, integrin αvβ,MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C,PDGF-Rα, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL,RON, RORI, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2,TGF-β, TRAIL-R, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1,VEGFR2, and vimentin.

The antigen can be associated with an inflammatory disease. Non-limitingexamples of antigens associated with inflammatory disease include, e.g.,AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154(CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (α chain of IL-2receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE Fc region, IL-1, IL-12,IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6receptor, integrin α4, integrin α4β7, LFA-1 (CD11a), myostatin, OX-40,scleroscin, SOST, TGF beta 1, TNF-α, and VEGF-A.

Where the first member of the specific binding pair is an antigen, thesecond member of the specific binding pair can be an antibody-basedscaffold (e.g., an antibody) or a non-antibody-based scaffold. In somecases, the second member of the specific binding pair is present on thesurface of a cell. In some cases, the second member of the specificbinding pair is immobilized on an insoluble support. In some cases, thesecond member of the specific binding pair is soluble. In some cases,the second member of the specific binding pair is present in anextracellular environment (e.g., extracellular matrix). In some cases,the second member of the specific binding pair is present in anartificial matrix. In some cases, the second member of the specificbinding pair is present in an acellular environment.

Targets of Non-Antibody-Based Recognition Scaffolds

In some cases, the first member of the specific binding pair is a targetof a non-antibody-based scaffold. Targets include, e.g., polypeptides,nucleic acids, glycoproteins, small molecules, carbohydrates, lipids,glycolipids, lipoproteins, lipopolysaccharides, etc.

Where the first member of the specific binding pair is a target of anon-antibody-based scaffold, the second member of the specific bindingpair is a non-antibody-based scaffold.

Receptors

In some cases, the first member of the specific binding pair is areceptor. In some cases, the receptor is a growth factor receptor. Insome cases, the receptor is a cytokine receptor. In some cases, thereceptor is a cell surface receptor that binds to a co-receptor on acell. In some cases, the receptor is a neurotransmitter receptor. Insome cases, the receptor binds to an extracellular matrix component. Insome cases, the receptor is an immunoglobulin Fc receptor.

Suitable receptors include, but are not limited to, a growth factorreceptor (e.g., a VEGF receptor); a killer cell lectin-like receptorsubfamily K, member 1 (NKG2D) polypeptide (receptor for MICA, MICB, andULB6); a cytokine receptor (e.g., an IL-13 receptor; an IL-2 receptor;etc.); an epidermal growth factor (EGF) receptor; Her2; CD27; a naturalcytotoxicity receptor (NCR) (e.g., NKP30 (NCR3/CD337) polypeptide(receptor for HLA-B-associated transcript 3 (BAT3) and B7-H6); etc.); aT cell antigen receptor; a dihydrofolate receptor; a chimeric cytokinereceptor; an Fc receptor; an extracellular matrix receptor (e.g. anintegrin); a cell adhesion receptor (e.g. a cadherin); animmunoregulatory receptor including both positive co-receptors (e.g.CD28) and negative (immunosuppressive) co-receptors (e.g., PD1); acytokine receptor; and a receptor for a immunoregulatory molecule (e.g.TGFP), etc. In some cases, the receptor is truncated, relative to thewild-type receptor.

Where the first member of the specific binding pair is a receptor, thesecond member of the specific binding pair is target of the receptor,where the target can be a ligand for the receptor, or a co-receptor. Insome cases, the second member of the specific binding pair is present onthe surface of a cell. In some cases, the second member of the specificbinding pair is immobilized on an insoluble support. In some cases, thesecond member of the specific binding pair is soluble. In some cases,the second member of the specific binding pair is present in anextracellular environment (e.g., extracellular matrix). In some cases,the second member of the specific binding pair is present in anartificial matrix. In some cases, the second member of the specificbinding pair is present in an acellular environment.

Notch Receptor Polypeptide

As noted above, a chimeric Notch receptor polypeptide of the presentdisclosure comprises a Notch receptor polypeptide having a length offrom 50 amino acids to 1000 amino acids and comprising one or moreligand-inducible proteolytic cleavage sites.

In some cases, the Notch receptor polypeptide present in a chimericNotch receptor polypeptide of the present disclosure has a length offrom 50 amino acids (aa) to 1000 aa, e.g., from 50 aa to 75 aa, from 75aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aato 250 aa, from 250 a to 300 aa, from 300 aa to 350 aa, from 350 aa to400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa, from 500 aa to 550aa, from 550 aa to 600 aa, from 600 aa to 650 aa, from 650 aa to 700 aa,from 700 aa to 750 aa, from 750 aa to 800 aa, from 800 aa to 850 aa,from 850 aa to 900 aa, from 900 aa to 950 aa, or from 950 aa to 1000 aa.In some cases, the Notch receptor polypeptide present in a chimericNotch receptor polypeptide of the present disclosure has a length offrom 300 aa to 400 aa. In some cases, the Notch receptor polypeptidepresent in a chimeric Notch receptor polypeptide of the presentdisclosure has a length of from 300 aa to 350 aa. In some cases, theNotch receptor polypeptide present in a chimeric Notch receptorpolypeptide of the present disclosure has a length of from 300 aa to 325aa. In some cases, the Notch receptor polypeptide present in a chimericNotch receptor polypeptide of the present disclosure has a length offrom 350 aa to 400 aa. In some cases, the Notch receptor polypeptidepresent in a chimeric Notch receptor polypeptide of the presentdisclosure has a length of from 750 aa to 850 aa. In some cases, theNotch receptor polypeptide present in a chimeric Notch receptorpolypeptide of the present disclosure has a length of from 50 aa to 75aa. In some cases, the Notch receptor polypeptide present in a chimericNotch receptor polypeptide of the present disclosure has a length offrom 310 aa to 320 aa, e.g., 310 aa, 311 aa, 312 aa, 313 aa, 314 aa, 315aa, 316 aa, 317 aa, 318 aa, 319 aa, or 320 aa. In some cases, the Notchreceptor polypeptide present in a chimeric Notch receptor polypeptide ofthe present disclosure has a length of 315 aa. In some cases, the Notchreceptor polypeptide present in a chimeric Notch receptor polypeptide ofthe present disclosure has a length of from 360 aa to 370 aa, e.g., 360aa, 361 aa, 362 aa, 363 aa 364 aa, 365 aa, 366 aa, 367 aa, 368 aa, 369aa, or 370 aa. In some cases, the Notch receptor polypeptide present ina chimeric Notch receptor polypeptide of the present disclosure has alength of 367 aa.

Notch Receptor Polypeptide Comprising a TM Domain

In some cases, the Notch receptor polypeptide present in a chimericNotch receptor polypeptide of the present disclosure comprises an aminoacid sequence having at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the following amino acid sequence:

(SEQ ID NO: 4) IPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRRQLCIQKL;where the TM domain is underlined; where the Notch receptor polypeptidecomprises an S2 proteolytic cleavage site and an S3 proteolytic cleavagesite; where the Notch receptor polypeptide has a length of from 50 aminoacids (aa) to 65 aa, e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, or 65 aa. In some cases, the Notch receptor polypeptidepresent in a chimeric Notch receptor polypeptide of the presentdisclosure comprises an amino acid sequence having at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the following aminoacid sequence:

(SEQ ID NO: 4) IPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRRQLCIQKL;where the TM domain is underlined; where the Notch receptor polypeptidecomprises an S2 proteolytic cleavage site and an S3 proteolytic cleavagesite; where the Notch receptor polypeptide has a length of 56 aminoacids.

Notch Receptor Polypeptide Comprising an LNR Segment, an HD-N Segment,an HD-C Segment, and a TM Domain

In some cases, the Notch receptor polypeptide present in a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: i) a LNR-A segment; ii) a LNR-B segment;iii) a LNR-C segment; iv) an HD-N segment, v) an HD-C segment; and vi) aTM domain. A LNR-A segment, LNR-B segment, and LNR-C segment cancollectively be referred to as an “LNR segment.” Such a Notch receptorpolypeptide is depicted schematically in FIG. 4A.

An LNR segment can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1442-1562 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and canhave a length of from 90 amino acids to 150 amino acids, e.g., from 90amino acids (aa) to 100 aa, from 100 aa to 110 aa, from 110 aa to 120aa, from 120 aa to 130 aa, from 130 aa to 140 aa, or from 140 aa to 150aa. In some cases, an LNR segment comprises an amino acid sequencehaving at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto amino acids 1442-1562 of the amino acid sequence depicted in FIG. 2A,or a corresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and hasa length of from 115 aa to 125 aa, e.g., 115, 116, 117, 118, 119, 120,121, 122, 123, 124, or 125 aa.

An LNR segment can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the following aminoacid sequence: PPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECE WDGLDC (SEQ IDNO:5); and can have a length of from 118 to 122 amino acids (e.g., 118,119, 120, 121, or 122 amino acids).

An HD-N segment can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1563-1664 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and canhave a length of from 90 amino acids (aa) to 110 aa, e.g., 90 aa to 95aa, 95 aa to 100 aa, 100 aa to 105 aa, or 105 aa to 110 aa. In somecases, an HD-N segment comprises an amino acid sequence having at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to amino acids1563-1664 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and hasa length of from 95 aa to 105 aa, e.g., 95, 96, 98, 98, 99, 100, 101,102, 103, 104, or 105 aa.

An HD-C segment can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1665-1733 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and canhave a length of from 60 amino acids (aa) to 80 aa, e.g., from 60 aa to65 aa, from 65 aa to 70 aa, from 70 aa to 75 aa, or from 75 aa to 80 aa.In some cases, an HD-C segment comprises an amino acid sequence havingat least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to aminoacids 1665-1733 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and hasa length of from 65 amino acids to 75 amino acids, e.g., 65, 66, 67, 68,69, 70, 71, 72, 73, 74, or 75 amino acids.

An HD segment (HD-N plus HD-C) can comprise an amino acid sequencehaving at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the following amino acid sequence:AAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLP (SEQ ID NO:6); and can have a lengthof 150, 151, 152, 153, or 154 amino acids.

A transmembrane segment can comprise an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to aminoacids 1736 to 1756 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and canhave a length of from 15 amino acids (aa) to 25 amino acids, e.g., 15,16, 17, 18, 29, 20, 21, 22, 23, 24, or 25 amino acids.

A transmembrane segment can comprise an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence: HLMYVAAAAFVLLFFVGCGVLLS (SEQ ID NO:7);and can have a length of 21, 22, 23, 24, or 25 amino acids.

In some cases, a Notch receptor polypeptide has a length of from about310 amino acids (aa) to about 320 aa (e.g., 310 aa, 311 aa, 312 aa, 313aa, 314 aa, 315 aa, 316 aa, 317 aa, 318 aa, 319 aa, or 320 aa), andcomprises an amino acid sequence having at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to amino acids 1442-1756 of the aminoacid sequence depicted in FIG. 2A, or a corresponding segment of anotherNotch receptor polypeptide, where examples of corresponding segments aredepicted in FIGS. 2B-2G.

In some cases, a Notch receptor polypeptide comprises an amino acidsequence having at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:PPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLS (SEQ ID NO:1); and has a length of from 300 amino acidsto 310 amino acids (e.g., 300, 301, 302, 303, 304, 305, 306, 307, 308,309, or 310 amino acids).

In some cases, a Notch receptor polypeptide comprises an amino acidsequence having at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVG CGVLLS (SEQID NO:2); and has a length of from 350 amino acids to 370 amino acids(e.g., 350 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362,363, 364, 365, 366, 367, 368, 369, or 370 amino acids).

Notch Receptor Polypeptide Comprising a Single EGF Repeat, an LNRSegment, an HD-N Segment, an HD-C Segment, and a TM Domain

In some cases, the Notch receptor polypeptide present in a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: i) a single EGF repeat; ii) an LNRsegment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain.Such a Notch receptor polypeptide is depicted schematically in FIG. 4B.

An EGF repeat can comprises an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids 1390 to1430 of the amino acid sequence depicted in FIG. 2A, or a correspondingsegment of another Notch receptor polypeptide, where examples ofcorresponding segments are depicted in FIGS. 2B-2G; and can have alength of from 35 amino acids (aa) to 45 aa (e.g., 35, 36, 37, 38, 39,40, 41, 42, 43, 44, or 45 aa).

An EGF repeat can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the followingsequence: PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCH (SEQ ID NO:8); and canhave a length of 35 amino acids to 40 amino acids (e.g., 35, 36, 37, 38,39, or 40 amino acids.

An LNR segment can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1442-1562 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and canhave a length of from 90 amino acids to 150 amino acids, e.g., from 90amino acids (aa) to 100 aa, from 100 aa to 110 aa, from 110 aa to 120aa, from 120 aa to 130 aa, from 130 aa to 140 aa, or from 140 aa to 150aa. In some cases, an LNR segment comprises an amino acid sequencehaving at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto amino acids 1442-1562 of the amino acid sequence depicted in FIG. 2A,or a corresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and hasa length of from 115 aa to 125 aa, e.g., 115, 116, 117, 118, 119, 120,121, 122, 123, 124, or 125 aa.

An LNR segment can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the following aminoacid sequence: PPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECE WDGLDC (SEQ IDNO:5); and can have a length of from 118 to 122 amino acids (e.g., 118,119, 120, 121, or 122 amino acids).

An HD-N segment can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1563-1664 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and canhave a length of from 90 amino acids (aa) to 110 aa, e.g., 90 aa to 95aa, 95 aa to 100 aa, 100 aa to 105 aa, or 105 aa to 110 aa. In somecases, an HD-N segment comprises an amino acid sequence having at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to amino acids1563-1664 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and hasa length of from 95 aa to 105 aa, e.g., 95, 96, 98, 98, 99, 100, 101,102, 103, 104, or 105 aa.

An HD-C segment can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1665-1733 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and canhave a length of from 60 amino acids (aa) to 80 aa, e.g., from 60 aa to65 aa, from 65 aa to 70 aa, from 70 aa to 75 aa, or from 75 aa to 80 aa.In some cases, an HD-C segment comprises an amino acid sequence havingat least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to aminoacids 1665-1733 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and hasa length of from 65 amino acids to 75 amino acids, e.g., 65, 66, 67, 68,69, 70, 71, 72, 73, 74, or 75 amino acids.

An HD segment (HD-N plus HD-C) can comprise an amino acid sequencehaving at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the following amino acid sequence:AAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLP (SEQ ID NO:6); and can have a lengthof 150, 151, 152, 153, or 154 amino acids.

A transmembrane segment can comprise an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to aminoacids 1736 to 1756 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and canhave a length of from 15 amino acids (aa) to 25 amino acids, e.g., 15,16, 17, 18, 29, 20, 21, 22, 23, 24, or 25 amino acids.

A transmembrane segment can comprise an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence: HLMYVAAAAFVLLFFVGCGVLLS (SEQ ID NO:7);and can have a length of 21, 22, 23, 24, or 25 amino acids.

In some cases, a Notch receptor polypeptide has a length of from about360 amino acids (aa) to about 375 aa (e.g., 360 aa, 361 aa, 362 aa, 363aa, 364 aa, 365 aa, 366 aa, 367 aa, 368 aa, 369 aa, 370 aa, 371 aa, 372aa, 373 aa, 374 aa, or 375 aa), and comprises an amino acid sequencehaving at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto amino acids 1390-1756 of the amino acid sequence depicted in FIG. 2A,or a corresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G.

In some cases, a Notch receptor polypeptide comprises a syntheticlinker. For example, in some cases, a Notch receptor polypeptidecomprises, in order from N-terminus to C-terminus: i) a syntheticlinker; ii) an EGF repeat; iii) an LNR segment; iv) an HD-N segment, v)an HD-C segment; and vi) a TM domain. Such a Notch receptor polypeptideis depicted schematically in FIG. 4C.

A synthetic linker can have a length of from about 10 amino acids (aa)to about 200 aa, e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 125 aa, from 125 aa to150 aa, from 150 aa to 175 aa, or from 175 aa to 200 aa. A syntheticlinker can have a length of from 10 aa to 30 aa, e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30aa. A synthetic linker can have a length of from 30 aa to 50 aa, e.g.,from 30 aa to 35 aa, from 35 aa to 40 aa, from 40 aa to 45 aa, or from45 aa to 50 aa.

In some instances, a synthetic linker, as described herein, may includean extracellular protein structural domain or a portion thereof.Extracellular protein structural domains suitable for use as a syntheticlinker include but are not limited to e.g., Ig-like extracellularstructural domains, Fc extracellular structural domains, fibronectinextracellular structural domains and the like. In some instances, asynthetic linker may include a plurality of extracellular proteinstructural domains where the plurality may include a plurality of thesame domain or a plurality of different domains.

Notch Receptor Polypeptide Comprising 2-11 EGF Repeats, an LNR Segment,an HD-N Segment, an HD-C Segment, and a TM Domain

In some cases, the Notch receptor polypeptide present in a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: i) from two to eleven EGF repeats; ii) anLNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TMdomain. Such a Notch receptor polypeptide is depicted schematically inFIG. 4D.

In some cases, the Notch receptor polypeptide present in a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: i) two EGF repeats; ii) an LNR segment;iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain. In somecases, the Notch receptor polypeptide present in a chimeric Notchreceptor polypeptide of the present disclosure comprises, in order fromN-terminus to C-terminus: i) three EGF repeats; ii) an LNR segment; iii)an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases,the Notch receptor polypeptide present in a chimeric Notch receptorpolypeptide of the present disclosure comprises, in order fromN-terminus to C-terminus: i) four EGF repeats; ii) an LNR segment; iii)an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases,the Notch receptor polypeptide present in a chimeric Notch receptorpolypeptide of the present disclosure comprises, in order fromN-terminus to C-terminus: i) five EGF repeats; ii) an LNR segment; iii)an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases,the Notch receptor polypeptide present in a chimeric Notch receptorpolypeptide of the present disclosure comprises, in order fromN-terminus to C-terminus: i) six EGF repeats; ii) an LNR segment; iii)an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases,the Notch receptor polypeptide present in a chimeric Notch receptorpolypeptide of the present disclosure comprises, in order fromN-terminus to C-terminus: i) seven EGF repeats; ii) an LNR segment; iii)an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases,the Notch receptor polypeptide present in a chimeric Notch receptorpolypeptide of the present disclosure comprises, in order fromN-terminus to C-terminus: i) eight EGF repeats; ii) an LNR segment; iii)an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases,the Notch receptor polypeptide present in a chimeric Notch receptorpolypeptide of the present disclosure comprises, in order fromN-terminus to C-terminus: i) nine EGF repeats; ii) an LNR segment; iii)an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases,the Notch receptor polypeptide present in a chimeric Notch receptorpolypeptide of the present disclosure comprises, in order fromN-terminus to C-terminus: i) ten EGF repeats; ii) an LNR segment; iii)an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases,the Notch receptor polypeptide present in a chimeric Notch receptorpolypeptide of the present disclosure comprises, in order fromN-terminus to C-terminus: i) eleven EGF repeats; ii) an LNR segment;iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain.

An EGF repeat can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids 1390 to1430 of the amino acid sequence depicted in FIG. 2A, or a correspondingsegment of another Notch receptor polypeptide, where examples ofcorresponding segments are depicted in FIGS. 2B-2G; and can have alength of from 35 amino acids (aa) to 45 aa (e.g., 35, 36, 37, 38, 39,40, 41, 42, 43, 44, or 45 aa).

An EGF repeat can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids 869-905(DINECVLSPCRHGASCQNTHGGYRCHCQAGYSGRNCE; SEQ ID NO:9) of the amino acidsequence depicted in FIG. 2A, or a corresponding segment of anotherNotch receptor polypeptide, where examples of corresponding segments aredepicted in FIGS. 2B-2G; and can have a length of from 35 amino acids toabout 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa).

An EGF repeat can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids 907-943(DIDDCRPNPCHNGGSCTDGINTAFCDCLPGFRGTFCE; SEQ ID NO:10) of the amino acidsequence depicted in FIG. 2A, or a corresponding segment of anotherNotch receptor polypeptide, where examples of corresponding segments aredepicted in FIGS. 2B-2G; and can have a length of from 35 amino acids toabout 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa).

An EGF repeat can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids 945-981(DINECASDPCRNGANCTDCVDSYTCTCPAGFSGIHCE; (SEQ ID NO:11) of the amino acidsequence depicted in FIG. 2A, or a corresponding segment of anotherNotch receptor polypeptide, where examples of corresponding segments aredepicted in FIGS. 2B-2G; and can have a length of from 35 amino acids toabout 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa).

An EGF repeat can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids 988-1019(TESSCFNGGTCVDGINSFTCLCPPGFTGSYCQ; SEQ ID NO:12) of the amino acidsequence depicted in FIG. 2A, or a corresponding segment of anotherNotch receptor polypeptide, where examples of corresponding segments aredepicted in FIGS. 2B-2G; and can have a length of from 30 amino acids(aa) to 35 aa (e.g., 30, 31, 32, 33, 34, or 35 aa).

An EGF repeat can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1021-1057 (DVNECDSQPCLHGGTCQDGCGSYRCTCPQGYTGPNCQ; SEQ ID NO:13) of theamino acid sequence depicted in FIG. 2A, or a corresponding segment ofanother Notch receptor polypeptide, where examples of correspondingsegments are depicted in FIGS. 2B-2G; and can have a length of from 35amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or40 aa).

An EGF repeat can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1064-1090 (DSSPCKNGGKCWQTHTQYRCECPSGWT; SEQ ID NO:14) of the amino acidsequence depicted in FIG. 2A, or a corresponding segment of anotherNotch receptor polypeptide, where examples of corresponding segments aredepicted in FIGS. 2B-2G; and can have a length of from 25 amino acids(aa) to 30 aa, e.g., 25, 26, 27, 28, 29, or 30 aa.

An EGF repeat can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1146-1180 (LVDECSPSPCQNGATCTDYLGGYSCKCVAGYHGVNC; SEQ ID NO:15) of theamino acid sequence depicted in FIG. 2A, or a corresponding segment ofanother Notch receptor polypeptide, where examples of correspondingsegments are depicted in FIGS. 2B-2G; and can have a length of from 35amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or40 aa).

An EGF repeat can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1184-1219 (IDECLSHPCQNGGTCLDLPNTYKCSCPRGTQGVHCE; SEQ ID NO:16) of theamino acid sequence depicted in FIG. 2A, or a corresponding segment ofanother Notch receptor polypeptide, where examples of correspondingsegments are depicted in FIGS. 2B-2G; and can have a length of from 35amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or40 aa).

An EGF repeat can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1238-1265 (CFNNGTCVDQVGGYSCTCPPGFVGERCE; SEQ ID NO:17) of the amino acidsequence depicted in FIG. 2A, or a corresponding segment of anotherNotch receptor polypeptide, where examples of corresponding segments aredepicted in FIGS. 2B-2G; and can have a length of from 25 amino acids(aa) to 30 aa, e.g., 25, 26, 27, 28, 29, or 30 aa.

An EGF repeat can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1267-1305 (DVNECLSNPCDARGTQNCVQRVNDFHCECRAGHTGRRCE; (SEQ ID NO: 18) ofthe amino acid sequence depicted in FIG. 2A, or a corresponding segmentof another Notch receptor polypeptide, where examples of correspondingsegments are depicted in FIGS. 2B-2G; and can have a length of from 35amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or40 aa).

An EGF repeat can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the followingsequence: PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCH (SEQ ID NO:8); and canhave a length of 35 amino acids to 40 amino acids (e.g., 35, 36, 37, 38,39, or 40 amino acids.

An LNR segment can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1442-1562 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and canhave a length of from 90 amino acids to 150 amino acids, e.g., from 90amino acids (aa) to 100 aa, from 100 aa to 110 aa, from 110 aa to 120aa, from 120 aa to 130 aa, from 130 aa to 140 aa, or from 140 aa to 150aa. In some cases, an LNR segment comprises an amino acid sequencehaving at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto amino acids 1442-1562 of the amino acid sequence depicted in FIG. 2A,or a corresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and hasa length of from 115 aa to 125 aa, e.g., 115, 116, 117, 118, 119, 120,121, 122, 123, 124, or 125 aa.

An LNR segment can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the following aminoacid sequence: PPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECE WDGLDC (SEQ IDNO:5); and can have a length of from 118 to 122 amino acids (e.g., 118,119, 120, 121, or 122 amino acids).

An HD-N segment can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1563-1664 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and canhave a length of from 90 amino acids (aa) to 110 aa, e.g., 90 aa to 95aa, 95 aa to 100 aa, 100 aa to 105 aa, or 105 aa to 110 aa. In somecases, an HD-N segment comprises an amino acid sequence having at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to amino acids1563-1664 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and hasa length of from 95 aa to 105 aa, e.g., 95, 96, 98, 98, 99, 100, 101,102, 103, 104, or 105 aa.

An HD-C segment can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1665-1733 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and canhave a length of from 60 amino acids (aa) to 80 aa, e.g., from 60 aa to65 aa, from 65 aa to 70 aa, from 70 aa to 75 aa, or from 75 aa to 80 aa.In some cases, an HD-C segment comprises an amino acid sequence havingat least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to aminoacids 1665-1733 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and hasa length of from 65 amino acids to 75 amino acids, e.g., 65, 66, 67, 68,69, 70, 71, 72, 73, 74, or 75 amino acids.

An HD segment (HD-N plus HD-C) can comprise an amino acid sequencehaving at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the following amino acid sequence:AAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLP (SEQ ID NO:6); and can have a lengthof 150, 151, 152, 153, or 154 amino acids.

A transmembrane segment can comprise an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to aminoacids 1736 to 1756 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and canhave a length of from 15 amino acids (aa) to 25 amino acids, e.g., 15,16, 17, 18, 29, 20, 21, 22, 23, 24, or 25 amino acids.

A transmembrane segment can comprise an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence: HLMYVAAAAFVLLFFVGCGVLLS (SEQ ID NO:7);and can have a length of 21, 22, 23, 24, or 25 amino acids.

In some cases, a Notch receptor polypeptide has a length of from about490 amino acids (aa) to about 900 aa, and comprises an amino acidsequence having at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to: i) amino acids 1267-1756; ii) 1238-1756; iii) 1184-1756;iv) 1146-1756; v) 1064-1756; vi) 1021-1756; vii) 988-1756; viii)945-1756; ix) 907-1756; or x) 869-1756, of the amino acid sequencedepicted in FIG. 2A, or a corresponding segment of another Notchreceptor polypeptide, where examples of corresponding segments aredepicted in FIGS. 2B-2G.

In some cases, a Notch receptor polypeptide comprises a syntheticlinker. For example, in some cases, a Notch receptor polypeptidecomprises, in order from N-terminus to C-terminus: i) two to eleven EGFrepeats; ii) a synthetic linker; iii) an LNR segment; iv) an HD-Nsegment, v) an HD-C segment; and vi) a TM domain. Such a Notch receptorpolypeptide is depicted schematically in FIG. 4E.

A synthetic linker can have a length of from about 10 amino acids (aa)to about 200 aa, e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 125 aa, from 125 aa to150 aa, from 150 aa to 175 aa, or from 175 aa to 200 aa. A syntheticlinker can have a length of from 10 aa to 30 aa, e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30aa. A synthetic linker can have a length of from 30 aa to 50 aa, e.g.,from 30 aa to 35 aa, from 35 aa to 40 aa, from 40 aa to 45 aa, or from45 aa to 50 aa.

Notch Receptor Polypeptide Comprising an HD-C Segment and a TM Domain

In some cases, a Notch receptor polypeptide comprises, in order fromN-terminus to C-terminus: i) an HD-C segment; and ii) a TM domain, wherethe Notch receptor polypeptide does not include an LNR segment. In somecases, the LNR segment is replaced with a heterologous polypeptide. Sucha Notch receptor polypeptide is depicted schematically in FIG. 4F.

An HD-C segment can comprise an amino acid sequence having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids1665-1733 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and canhave a length of from 60 amino acids (aa) to 80 aa, e.g., from 60 aa to65 aa, from 65 aa to 70 aa, from 70 aa to 75 aa, or from 75 aa to 80 aa.In some cases, an HD-C segment comprises an amino acid sequence havingat least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to aminoacids 1665-1733 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and hasa length of from 65 amino acids to 75 amino acids, e.g., 65, 66, 67, 68,69, 70, 71, 72, 73, 74, or 75 amino acids.

A transmembrane segment can comprise an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to aminoacids 1736 to 1756 of the amino acid sequence depicted in FIG. 2A, or acorresponding segment of another Notch receptor polypeptide, whereexamples of corresponding segments are depicted in FIGS. 2B-2G; and canhave a length of from 15 amino acids (aa) to 25 amino acids, e.g., 15,16, 17, 18, 29, 20, 21, 22, 23, 24, or 25 amino acids.

A transmembrane segment can comprise an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence: HLMYVAAAAFVLLFFVGCGVLLS (SEQ ID NO:7);and can have a length of 21, 22, 23, 24, or 25 amino acids.

In some cases, a Notch receptor polypeptide comprises an amino acidsequence having at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to amino acids 1665 to 1756 of the amino acid sequence depictedin FIG. 2A, or a corresponding segment of another Notch receptorpolypeptide, where examples of corresponding segments are depicted inFIGS. 2B-2G; and has a length of from 85 amino acids (aa) to 95 aa(e.g., 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 aa).

In some cases, a Notch receptor polypeptide comprises an amino acidsequence having at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to amino acids 1665 to 1756 of the amino acid sequence depictedin FIG. 2A, or a corresponding segment of another Notch receptorpolypeptide, where examples of corresponding segments are depicted inFIGS. 2B-2G; and comprises a heterologous polypeptide fused in-frame atthe N-terminus of the Notch receptor polypeptide.

Ligand-Inducible Proteolytic Cleavage Sites

As noted above, a chimeric Notch receptor polypeptide of the presentdisclosure comprises a Notch receptor polypeptide having a length offrom 50 amino acids to 1000 amino acids, and comprising one or moreligand-inducible proteolytic cleavage sites. As discussed above, achimeric Notch receptor polypeptide of the present disclosure comprises:a) an extracellular domain comprising a first member of a specificbinding pair; b) a Notch receptor polypeptide having a length of from 50amino acids to 1000 amino acids, and comprising one or moreligand-inducible proteolytic cleavage sites; and c) an intracellulardomain, where binding of the first member of the specific binding pairto a second member of a specific binding pair induces cleavage of theNotch receptor polypeptide at the one or more ligand-inducibleproteolytic cleavage sites, thereby releasing the intracellular domain.The second member (“ligand”) of the specific binding pair can be presenton a contacting (e.g., “sending”) cell.

In some cases, the Notch receptor polypeptide includes only oneligand-inducible proteolytic cleavage site. In some cases, the Notchreceptor polypeptide includes two ligand-inducible proteolytic cleavagesites. In some cases, the Notch receptor polypeptide includes threeligand-inducible proteolytic cleavage sites. For simplicity,ligand-inducible cleavage sites will be referred to herein as “S1,”“S2,” and “S3” ligand-inducible proteolytic cleavage sites.

In some cases, the Notch receptor polypeptide includes an S1ligand-inducible proteolytic cleavage site. An S1 ligand-inducibleproteolytic cleavage site can be located between the HD-N segment andthe HD-C segment. In some cases, the S1 ligand-inducible proteolyticcleavage site is a furin-like protease cleavage site. A furin-likeprotease cleavage site can have the canonical sequenceArg-X-(Arg/Lys)-Arg, where X is any amino acid; the protease cleavesimmediately C-terminal to the canonical sequence. For example, in somecases, an amino acid sequence comprising an S1 ligand-inducibleproteolytic cleavage site can have the amino acid sequence GRRRRELDPM(SEQ ID NO:19), where cleavage occurs between the “RE” sequence. Asanother example, an amino acid sequence comprising an S1ligand-inducible proteolytic cleavage site can have the amino acidsequence RQRRELDPM (SEQ ID NO:20), where cleavage occurs between the“RE” sequence.

In some cases, the Notch receptor polypeptide includes an S2ligand-inducible proteolytic cleavage site. An S2 ligand-inducibleproteolytic cleavage site can be located within the HD-C segment. Insome cases, the S2 ligand-inducible proteolytic cleavage site is anADAM-17-type protease cleavage site. An ADAM-17-type protease cleavagesite can comprise an Ala-Val dipeptide sequence, where the enzymecleaves between the Ala and the Val. For example, in some cases, aminoacid sequence comprising an S2 ligand-inducible proteolytic cleavagesite can have the amino acid sequence KIEAVKSE (SEQ ID NO:21), wherecleavage occurs between the “AV” sequence. As another example, an aminoacid sequence comprising an S2 ligand-inducible proteolytic cleavagesite can have the amino acid sequence KIEAVQSE (SEQ ID NO:22), wherecleavage occurs between the “AV” sequence.

In some cases, the Notch receptor polypeptide includes an S3ligand-inducible proteolytic cleavage site. An S3 ligand-inducibleproteolytic cleavage site can be located within the TM domain. In somecases, the S3 ligand-inducible proteolytic cleavage site is agamma-secretase (γ-secretase) cleavage site. A γ-secretase cleavage sitecan comprise a Gly-Val dipeptide sequence, where the enzyme cleavesbetween the Gly and the Val. For example, in some cases, an S3ligand-inducible proteolytic cleavage site has the amino acid sequenceVGCGVLLS (SEQ ID NO:23), where cleavage occurs between the “GV”sequence. In some cases, an S3 ligand-inducible proteolytic cleavagesite comprises the amino acid sequence GCGVLLS (SEQ ID NO:24).

In some cases, the Notch receptor polypeptide lacks an S1ligand-inducible proteolytic cleavage site. In some cases, the Notchreceptor polypeptide lacks an S2 ligand-inducible proteolytic cleavagesite. In some cases, the Notch receptor polypeptide lacks an S3ligand-inducible proteolytic cleavage site. In some cases, the Notchreceptor polypeptide lacks both an S1 ligand-inducible proteolyticcleavage site and an S2 ligand-inducible proteolytic cleavage site. Insome cases, the Notch receptor polypeptide includes an S3ligand-inducible proteolytic cleavage site; and lacks both an S1ligand-inducible proteolytic cleavage site and an S2 ligand-inducibleproteolytic cleavage site. Examples are depicted schematically in FIG.4G.

Intracellular Domain

As noted above, a chimeric Notch receptor polypeptide of the presentdisclosure comprises an intracellular domain that is released followingbinding of the chimeric Notch receptor polypeptide to the second memberof the specific binding pair, where binding of the chimeric Notchreceptor polypeptide to the second member of the specific binding pairinduces cleavage of an above-mentioned proteolytic cleavage site.

The intracellular domain comprises an amino acid sequence that isheterologous to the Notch receptor polypeptide. In other words, theintracellular domain comprises an amino acid sequence that is notnaturally present in a Notch receptor polypeptide.

The intracellular domain, when released from the chimeric Notch receptorpolypeptide, provides an effector function, where effector functionsinclude, e.g., increased production of one or more cytokines by thecell; reduced production of one or more cytokines by the cell; increasedor decreased production of a hormone by the cell; production of anantibody by the cell; a change in organelle activity; a change intrafficking of a polypeptide within the cell; a change in transcriptionof a target gene; a change in activity of a protein; a change in cellbehavior, e.g., cell death; cellular proliferation; effects on cellulardifferentiation; effects on cell survival; modulation of cellularsignaling responses; etc. In some cases, the intracellular domain, whenreleased from the chimeric Notch receptor polypeptide, provides for achange in transcription of a target gene. In some cases, theintracellular domain, when released from the chimeric Notch receptorpolypeptide, provides for an increase in the transcription of a targetgene. In some cases, the intracellular domain, when released from thechimeric Notch receptor polypeptide, provides for a decrease in a targetgene.

The intracellular domain can be any of a wide variety of polypeptides,where examples include, but are not limited to, transcriptionalactivators; transcriptional repressors; transcriptional co-activators;transcriptional co-repressors; DNA binding polypeptides; RNA bindingpolypeptides; translational regulatory polypeptides; hormones;cytokines; toxins; antibodies; chromatin modulators; suicide proteins;organelle specific polypeptides (e.g., a nuclear pore regulator, amitochondrial regulator, an endoplasmic reticulum regulator, and thelike); pro-apoptosis polypeptides; anti-apoptosis polypeptides; otherpolypeptides that promote cell death through other mechanisms;pro-proliferation polypeptides; anti-proliferative polypeptides; immuneco-stimulatory polypeptides; site-specific nucleases; recombinases;inhibitory immunoreceptors; an activating immunoreceptor; Cas9 andvariants of RNA targeted nucleases; and DNA recognition polypeptides;dominant negative variants of a polypeptide; a signaling polypeptide; areceptor tyrosine kinase; a non-receptor tyrosine kinase; a polypeptidethat promotes differentiation; and the like.

In some cases, the intracellular domain comprises a signalingpolypeptide. Suitable signaling polypeptides include, e.g., STAT3/5,Akt, Myc, and the like. In some cases, the signaling polypeptide is apart of a PI3K/mTOR-, NFκB-, MAPK-, STAT-, FAK-, MYC, or TGF-β mediatedsignaling pathway. In some cases, the signaling polypeptide is a part ofa Ras/Raf/Mek/Erk1/2, a JAK/STAT3, or a PI3K/Akt signaling pathway.

In some cases, the intracellular domain comprises dominant negativevariant of a polypeptide, e.g., a dominant negative variant of asignaling polypeptide. Examples of dominant negative variants include,e.g., a dominant negative TGF-β receptor; a dominant negative variant ofSTAT3 comprising one or more mutations affecting the DNA binding domainof STAT3 that functions as a dominant negative variant; and the like.

In some cases, the intracellular domain is an antibody-based scaffold ora non-antibody-based scaffold that blocks or alters a cellular activitywhen released from the chimeric Notch receptor polypeptide.

In some cases, the intracellular domain comprises an immunoreceptor,e.g., an activating immunoreceptor or an inhibitory immunoreceptor. Asuitable activating immunoreceptor can comprise an immunoreceptortyrosine-based activation motif (ITAM). An ITAM motif is YX₁X₂L/I, whereX₁ and X₂ are independently any amino acid. A suitable intracellularsignaling domain can be an ITAM motif-containing portion that is derivedfrom a polypeptide that contains an ITAM motif. For example, a suitableintracellular signaling domain can be an ITAM motif-containing domainfrom any ITAM motif-containing protein. Thus, a suitable intracellularsignaling domain need not contain the entire sequence of the entireprotein from which it is derived. Examples of suitable ITAMmotif-containing polypeptides include, but are not limited to: DAP12;FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3epsilon); CD3G (CD3 gamma); CD3Z (CD3 zeta); and CD79A (antigen receptorcomplex-associated protein alpha chain). As one non-limiting example, asuitable ITAM motif-containing polypeptide can comprise an amino acidsequence having at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, or100%, amino acid sequence identity to the following amino acid sequence:ESPYQELQGQRSDVYSDLNTQ (SEQ ID NO:25), where the ITAM motifs are in boldand are underlined. As another example, a suitable ITAM motif-containingpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to the following amino acid sequence: DGVYTGLSTRNQETYETLKHE(SEQ ID NO:26), where the ITAM motifs are in bold and are underlined.The polypeptide can comprise an ITAM motif-containing portion of thefull length CD3 zeta amino acid sequence. As another example, a suitableITAM motif-containing polypeptide can comprise an amino acid sequencehaving at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, or 100%, aminoacid sequence identity to any of the following amino acid sequences:RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:27);NQLYNELNLGRREEYDVLDKR (SEQ ID NO:28); EGLYNELQKDKMAEAYSEIGMK (SEQ ID NO:29); or DGLYQGLSTATKDTYDALHMQ (SEQ ID NO:30), where the ITAM motifs arein bold and are underlined.

Intracellular signaling domains suitable for use in a chimeric Notchpolypeptide of the present disclosure include immunoreceptortyrosine-based activation motif (ITAM)-containing intracellularsignaling polypeptides. An ITAM motif is YX₁X2L/I, where X₁ and X₂ areindependently any amino acid. In some cases, the intracellular signalingdomain of a chimeric Notch polypeptide comprises 1, 2, 3, 4, or 5 ITAMmotifs. In some cases, an ITAM motif is repeated twice in anintracellular signaling domain, where the first and second instances ofthe ITAM motif are separated from one another by 6 to 8 amino acids,e.g., (YX₁X₂L/I)(X₃)(YX₁X₂L/I), where n is an integer from 6 to 8, andeach of the 6-8 X₃ can be any amino acid. In some cases, theintracellular signaling domain of a chimeric Notch polypeptide comprises3 ITAM motifs.

A suitable intracellular signaling domain can be an ITAMmotif-containing portion that is derived from a polypeptide thatcontains an ITAM motif. For example, a suitable intracellular signalingdomain can be an ITAM motif-containing domain from any ITAMmotif-containing protein. Thus, a suitable intracellular signalingdomain need not contain the entire sequence of the entire protein fromwhich it is derived. Examples of suitable ITAM motif-containingpolypeptides include, but are not limited to: DAP12; FCER1G (Fc epsilonreceptor 1 gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3gamma); CD3Z (CD3 zeta); and CD79A (antigen receptor complex-associatedprotein alpha chain).

In some cases, the intracellular signaling domain is derived from DAP12(also known as TYROBP; TYRO protein tyrosine kinase binding protein;KARAP; PLOSL; DNAX-activation protein 12; KAR-associated protein; TYROprotein tyrosine kinase-binding protein; killer activating receptorassociated protein; killer-activating receptor-associated protein;etc.). For example, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to any of the following amino acid sequences (4 isoforms):MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO:31);MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO:32);MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO:33); orMGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO:34), where the ITAMmotifs are in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide cancomprise an ITAM motif-containing portion of the full length DAP12 aminoacid sequence. Thus, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to the following amino acid sequence: ESPYQELQGQRSDVYSDLNTQ(SEQ ID NO:25), where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from FCER1G(also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptorgamma-chain; fc-epsilon RI-gamma; fcRgamma; fceRI gamma; high affinityimmunoglobulin epsilon receptor subunit gamma; immunoglobulin Ereceptor, high affinity, gamma chain; etc.). For example, a suitableintracellular signaling domain polypeptide can comprise an amino acidsequence having at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, or 100%amino acid sequence identity to the following amino acid sequence:MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO:35), where the ITAM motifs are inbold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide cancomprise an ITAM motif-containing portion of the full length FCER1Gamino acid sequence. Thus, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to the following amino acid sequence: DGVYTGLSTRNQETYETLKHE(SEQ ID NO:26), where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from T-cellsurface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA;T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, deltapolypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 deltachain; T-cell surface glycoprotein CD3 delta chain; etc.). For example,a suitable intracellular signaling domain polypeptide can comprise anamino acid sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, or 100%, amino acid sequence identity to a contiguous stretch offrom about 100 amino acids to about 110 amino acids (aa), from about 110aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aato about 130 aa, from about 130 aa to about 140 aa, from about 140 aa toabout 150 aa, or from about 150 aa to about 170 aa, of either of thefollowing amino acid sequences (2 isoforms):MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO:36) orMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLRNDQVYQPLRDRDDAQYSHL GGNWARNK (SEQID NO:37), where the ITAM motifs are in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD3 deltaamino acid sequence. Thus, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to the following amino acid sequence: DQVYQPLRDRDDAQYSHLGGN(SEQ ID NO:38), where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from T-cellsurface glycoprotein CD3 epsilon chain (also known as CD3e, T-cellsurface antigen T3/Leu-4 epsilon chain, T-cell surface glycoprotein CD3epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.). For example, asuitable intracellular signaling domain polypeptide can comprise anamino acid sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, or 100%, amino acid sequence identity to a contiguous stretch offrom about 100 amino acids to about 110 amino acids (aa), from about 110aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aato about 130 aa, from about 130 aa to about 140 aa, from about 140 aa toabout 150 aa, or from about 150 aa to about 205 aa, of the followingamino acid sequence:MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:39), where the ITAM motifsare in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD3 epsilonamino acid sequence. Thus, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to the following amino acid sequence: NPDYEPIRKGQRDLYSGLNQR(SEQ ID NO:40), where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from T-cellsurface glycoprotein CD3 gamma chain (also known as CD3G, T-cellreceptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3complex), etc.). For example, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to a contiguous stretch of from about 100 amino acids to about110 amino acids (aa), from about 110 aa to about 115 aa, from about 115aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aato about 140 aa, from about 140 aa to about 150 aa, or from about 150 aato about 180 aa, of the following amino acid sequence:MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQ GNQLRRN (SEQID NO:41), where the ITAM motifs are in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD3 gammaamino acid sequence. Thus, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to the following amino acid sequence: DQLYQPLKDREDDQYSHLQGN(SEQ ID NO:42), where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from T-cellsurface glycoprotein CD3 zeta chain (also known as CD3Z, T-cell receptorT3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). Forexample, a suitable intracellular signaling domain polypeptide cancomprise an amino acid sequence having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, or 100%, amino acid sequence identity to acontiguous stretch of from about 100 amino acids to about 110 aminoacids (aa), from about 110 aa to about 115 aa, from about 115 aa toabout 120 aa, from about 120 aa to about 130 aa, from about 130 aa toabout 140 aa, from about 140 aa to about 150 aa, or from about 150 aa toabout 160 aa, of either of the following amino acid sequences (2isoforms): MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:43) orMKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:44), where theITAM motifs are in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD3 zetaamino acid sequence. Thus, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to any of the following amino acid sequences:RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:27);NQLYNELNLGRREEYDVLDKR (SEQ ID NO:28); EGLYNELQKDKMAEAYSEIGMK (SEQ IDNO:29); or DGLYQGLSTATKDTYDALHMQ (SEQ ID NO:30), where the ITAM motifsare in bold and are underlined.

In some cases, the intracellular signaling domain is derived from CD79A(also known as B-cell antigen receptor complex-associated protein alphachain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membraneglycoprotein; ig-alpha; membrane-bound immunoglobulin-associatedprotein; surface IgM-associated protein; etc.). For example, a suitableintracellular signaling domain polypeptide can comprise an amino acidsequence having at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, or100%, amino acid sequence identity to a contiguous stretch of from about100 amino acids to about 110 amino acids (aa), from about 110 aa toabout 115 aa, from about 115 aa to about 120 aa, from about 120 aa toabout 130 aa, from about 130 aa to about 150 aa, from about 150 aa toabout 200 aa, or from about 200 aa to about 220 aa, of either of thefollowing amino acid sequences (2 isoforms):

MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO:45);or

MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO:46), where the ITAM motifs are in bold andare underlined.

Likewise, a suitable intracellular signaling domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD79A aminoacid sequence. Thus, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to the following amino acid sequence: ENLYEGLNLDDCSMYEDISRG(SEQ ID NO:47), where the ITAM motifs are in bold and are underlined.

DAP10/CD28

Intracellular signaling domains suitable for use in a chimeric Notchpolypeptide of the present disclosure include a DAP10/CD28 typesignaling chain.

An example of a DAP10 signaling chain is the amino acid sequence is:RPRRSPAQDGKVYINMPGRG (SEQ ID NO:48). In some embodiments, a suitableintracellular signaling domain comprises an amino acid sequence havingat least about 85%, at least about 90%, at least about 95%, at leastabout 98%, or at least about 99%, amino acid sequence identity to theentire length of the amino acid sequence RPRRSPAQDGKVYINMPGRG (SEQ IDNO:48).

An example of a CD28 signaling chain is the amino acid sequence isFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRS(SEQ ID NO:49). In some embodiments, a suitable intracellular signalingdomain comprises an amino acid sequence having at least about 85%, atleast about 90%, at least about 95%, at least about 98%, or at leastabout 99%, amino acid sequence identity to the entire length of theamino acid sequence

(SEQ ID NO: 49) FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSD YMNM TPRRPGPTRKHYQPYAPPRDFAAYRS.

ZAP70

Intracellular signaling domains suitable for use in a chimeric Notchpolypeptide of the present disclosure include a ZAP70 polypeptide, e.g.,a polypeptide comprising an amino acid sequence having at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to a contiguousstretch of from about 300 amino acids to about 400 amino acids, fromabout 400 amino acids to about 500 amino acids, or from about 500 aminoacids to 619 amino acids, of the following amino acid sequence:

(SEQ ID NO: 50) MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSL ASKVEGPPGSTQKAEAACA.

Co-stimulatory domains derived from receptors are suitable for use asthe intracellular domain of a chimeric Notch polypeptide of the presentdisclosure. The co-stimulatory domain can be an intracellular portion ofa transmembrane protein (i.e., the co-stimulatory domain can be derivedfrom a transmembrane protein). Non-limiting examples of suitableco-stimulatory polypeptides include, but are not limited to, 4-1BB(CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD28 (also known asTp44). For example, a suitable co-stimulatory domain can comprise anamino acid sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, or 100% amino acid sequence identity to the following amino acidsequence:

(SEQ ID NO: 51) FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein 4-1BB (also known asTNFRSF9; CD137; 4-1BB; CDw137; ILA; etc.). For example, a suitableco-stimulatory domain can comprise an amino acid sequence having atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, or 100% amino acid sequenceidentity to the following amino acid sequence:

(SEQ ID NO: 52) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein OX-40 (also known asTNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, TXGP1L). For example, asuitable co-stimulatory domain can comprise an amino acid sequencehaving at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, or 100% aminoacid sequence identity to the following amino acid sequence:RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:53).

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein BTLA (also known asBTLA1 and CD272). For example, a suitable co-stimulatory domain cancomprise an amino acid sequence having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, or 100% amino acid sequence identity to thefollowing amino acid sequence:

(SEQ ID NO: 54) CCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNV KEAPTEYASICVRS.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD27 (also known asS152, T14, TNFRSF7, and Tp55). For example, a suitable co-stimulatorydomain can comprise an amino acid sequence having at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, or 100% amino acid sequence identity to thefollowing amino acid sequence:

(SEQ ID NO: 55) HQRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD30 (also known asTNFRSF8, D1S166E, and Ki-1). For example, a suitable co-stimulatorydomain can comprise an amino acid sequence having at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, or 100% amino acid sequence identity to acontiguous stretch of from about 100 amino acids to about 110 aminoacids (aa), from about 110 aa to about 115 aa, from about 115 aa toabout 120 aa, from about 120 aa to about 130 aa, from about 130 aa toabout 140 aa, from about 140 aa to about 150 aa, from about 150 aa toabout 160 aa, or from about 160 aa to about 185 aa of the followingamino acid sequence:

(SEQ ID NO: 56) RRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein GITR (also known asTNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a suitableco-stimulatory domain can comprise an amino acid sequence having atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, or 100% amino acid sequenceidentity to the following amino acid sequence:

(SEQ ID NO: 57) HIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLG DLWV.

In some cases, the co-stimulatory domain derived from an intracellularportion of the transmembrane protein HVEM (also known as TNFRSF14,RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2). For example, asuitable co-stimulatory domain can comprise an amino acid sequencehaving at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, or 100% aminoacid sequence identity to the following amino acid sequence:

(SEQ ID NO: 58) CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH.

A suitable inhibitory immunoreceptor can comprise an immunoreceptortyrosine-based inhibition motif (ITIM), an immunoreceptor tyrosine-basedswitch motif (ITSM), an NpxY motif, or a YXXΦ motif. Suitable inhibitorimmunoreceptors include PD1; CTLA4; BTLA; CD160; KRLG-1; 2B4; Lag-3; andTim-3. See, e.g., Odorizzi and Wherry (2012) J. Immunol. 188:2957; andBaitsch et al. (2012) PLoSOne 7:e30852.

In some cases, a suitable inhibitory immunoreceptor comprises an aminoacid sequence having at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the following PD1 amino acid sequence:

(SEQ ID NO: 59) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEV PTAHPSPSPRPAGQFQTLV.

In some cases, a suitable inhibitory immunoreceptor comprises an aminoacid sequence having at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the following CTLA4 amino acid sequence:

(SEQ ID NO: 60) MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN.

In some cases, a suitable inhibitory immunoreceptor comprises an aminoacid sequence having at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the following CD160 amino acid sequence:

(SEQ ID NO: 61) MLLEPGRGCCALAILLAIVDIQSGGCINITSSASQEGTRLNLICTVWHKKEEAEGFVVFLCKDRSGDCSPETSLKQLRLKRDPGIDGVGEISSQLMFTISQVTPLHSGTYQCCARSQKSGIRLQGHFFSILFTETGNYTVTGLKQRQHLEFSHNEGTLSSGFLQEKVWVMLVTSLVALQAL.

In some cases, a suitable inhibitory immunoreceptor comprises an aminoacid sequence having at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the following T-cell immunoglobulin and mucindomain-3 (Tim-3) amino acid sequence:

(SEQ ID NO: 62) MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTLQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAM P.

In some cases, a suitable inhibitory immunoreceptor comprises an aminoacid sequence having at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to amino acids 23-525 of the following lymphocyteactivation gene 3 (Lag-3) amino acid sequence:

(SEQ ID NO: 63) MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL.

In some cases, the intracellular domain is a Siglec. See, e.g., Varkiand Angata (2006) Glycobiol. 16:1R. In some cases, the Siglec isSiglec-15. In some cases, the intracellular domain is KIR2DL4. Miah etal. (2008) J. Immunol. 180:2922.

In some cases, the intracellular domain is a recombinase. Suitablerecombinases include a Cre recombinase; a Flp recombinase; a Drerecombinase; and the like. A suitable recombinase is a FLPe recombinase(see, e.g., Akbudak and Srivastava (2011) Mol. Biotechnol. 49:82). Asuitable recombinase is a Flpo recombinase.

A recombinase, as described herein, may be an intact recombinase or asplit recombinase. Portions of a split recombinase may be expressed fromthe same or different expression constructs. In some instances, twoparts of a split recombinase may be operably linked to differentbinding-triggered transcriptional switches. In other instances, a firstpart of a split recombinase may be operably linked to a bindingtriggered transcriptional switch and the second part of the splitrecombinase may be separately expressed from an expression construct.

Where split recombinases are utilized, e.g., as in logic gated SynNotchcircuits, the portions of the split recombinase may be arranged in andexpressed from one or more expression cassettes with other components invarious ways essentially as described below regarding splittranscription factors.

Accordingly, activation of one or more binding-triggered transcriptionalswitches may induce expression of portions of split recombinasesresulting in heterodimerization and/or complex formation of the splitrecombinase portions resulting in formation of a functional recombinase.Alternatively, activation of one or more binding-triggeredtranscriptional switches may result in release of recombinase portionsfrom the one or more binding-triggered transcriptional switchesresulting in heterodimerization and/or complex formation of the splitrecombinase portions resulting in formation of a functional recombinase.In addition, induction and release of split recombinase portions may becombined, e.g., where activation of one or more binding-triggeredtranscriptional switches may induce expression of portions of splitrecombinases and release of split recombinase portions from the one ormore binding-triggered transcriptional switches resulting inheterodimerization and/or complex formation of the split recombinaseportions resulting in formation of a functional recombinase.

Suitable split recombinases include but are not limited to e.g., splitCre recombinase as described in e.g., Beckervordersandforth R et al.,Stem Cell Reports. 2014; 2(2):153-62 Wen M et al., PLoS One. 2014;9(10):e110290 O'Brien S P et al., Biotechnol J. 2014; 9(3):355-61 Wang Pet al., Sci Rep. 2012; 2:497 Hirrlinger J et al., PLoS One. 2009;4(12):e8354 Hirrlinger J et al., PLoS One. 2009; 4(1):e4286; thedisclosures of which are incorporated herein by reference in theirentirety.

A suitable Cre recombinase can comprise an amino acid sequence having atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, or 100%, amino acidsequence identity to the following amino acid sequence:VSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGD (SEQ ID NO:64); andcan have a length of from 335 amino acids (aa) to 350 aa.

A suitable FLPe recombinase can comprise an amino acid sequence havingat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or 100%, amino acidsequence identity to the following amino acid sequence:MSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAAELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRYPAWNGIISQEVLDYLSSYINRRIGPVEQKLISEEDL (SEQ ID NO:65); and can have a length of from 430 aminoacids to 445 amino acids.

Suitable site-specific nucleases include, but are not limited to, anRNA-guided DNA binding protein having nuclease activity, e.g., a Cas9polypeptide; a transcription activator-like effector nuclease (TALEN);Zinc-finger nucleases; and the like.

Cas9 polypeptides are known in the art; see, e.g., Fonfara et al. (2014)Nucl. Acids Res. 42:2577; and Sander and Joung (2014) Nat. Biotechnol.32:347. A Cas9 polypeptide can comprise an amino acid sequence having atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, or 100%, amino acidsequence identity to the amino acid sequence depicted in FIG. 36.

In some cases, the intracellular domain is a Cas9 variant that lacksnuclease activity, but retains DNA target-binding activity. Such a Cas9variant is referred to herein as a “dead Cas9” or “dCas9.” See, e.g., Qiet al. (2013) Cell 152:1173. A dCas9 polypeptide can comprise a D10Aand/or an H840A amino acid substitution of the amino acid sequencedepicted in FIG. 36 or corresponding amino acids in another Cas9polypeptide.

In some cases, the intracellular domain is a chimeric dCas9, e.g., afusion protein comprising dCas9 and a fusion partner, where suitablefusion partners include, e.g., a non-Cas9 enzyme that provides for anenzymatic activity, where the enzymatic activity is methyltransferaseactivity, demethylase activity, acetyltransferase activity, deacetylaseactivity, kinase activity, phosphatase activity, ubiquitin ligaseactivity, deubiquitinating activity, adenylation activity, deadenylationactivity, SUMOylating activity, deSUMOylating activity, ribosylationactivity, deribosylation activity, myristoylation activity ordemyristoylation activity. In some cases, the intracellular domain is achimeric dCas9, e.g., a fusion protein comprising dCas9 and a fusionpartner, where suitable fusion partners include, e.g., a non-Cas9 enzymethat provides for an enzymatic activity, where the enzymatic activity isnuclease activity, methyltransferase activity, demethylase activity, DNArepair activity, DNA damage activity, deamination activity, dismutaseactivity, alkylation activity, depurination activity, oxidationactivity, pyrimidine dimer forming activity, integrase activity,transposase activity, recombinase activity, polymerase activity, ligaseactivity, helicase activity, photolyase activity or glycosylaseactivity.

In some cases, the intracellular domain is a chimeric dCas9, e.g., afusion protein comprising dCas9 and a fusion partner, where suitablefusion partners include, e.g., transcription activator or atranscription repressor domain (e.g., the Kruppel associated box (KRABor SKD); the Mad mSIN3 interaction domain (SID); the ERF repressordomain (ERD), etc.); zinc-finger-based artificial transcription factors(see, e.g., Sera (2009) Adv. Drug Deliv. 61:513); TALE-based artificialtranscription factors (see, e.g., Liu et al. (2013) Nat. Rev. Genetics14:781); and the like.

In some cases, the intracellular domain is an apoptosis inducer. Asuitable apoptosis inducer includes tBID. The term “tBID” refers to theC-terminal truncated fragment of the BH3 interacting death agonist (BID)protein which results from the enzymatic cleavage of cytosolic BID(e.g., by active caspase). At an early stage of apoptosis, tBIDtranslocates to the mitochondria and mediates the release of Cyt ctherefrom. Non-limiting examples of tBID proteins include human tBID(amino acids 61-195 of the amino acid sequence provided in GenBankAccession No. CAG30275).

Human tBID has the following amino acid sequence:gnrsshsrlgrieadsesgediirniarhlagvgdsmdrsippglvnglaedrnrdlataleqllqayprdmekektmlvlalllakkvashtpsllrdvfhttvnfingnlrtyvrslarngmd (SEQ ID NO:66).

In some embodiments, the intracellular domain comprises an amino acidsequence having at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the human tBID amino acid sequence provided above; and has alength of from about 120 amino acids (aa) to 150 aa, e.g., from 120 aato 125 aa, from 125 aa to 130 aa, from 130 aa to 135 aa, from 135 aa to140 aa, from 140 aa to 145 aa, or from 145 aa to 150 aa. In some cases,the intracellular domain comprises an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to thehuman tBID amino acid sequence provided above; and has a length of 135aa.

In some cases, the intracellular domain is a transcription factor.Examples of suitable transcription factors are those presented in Table1 of U.S. Patent Application No. 2014/0308746. Non-limiting examples ofsuitable transcription factors are depicted in FIGS. 37-66. Non-limitingexamples of suitable transcriptional activators and transcriptionalrepressors are depicted in FIGS. 37-83. In some cases, the intracellulardomain is a transcriptional regulator. Non-limiting examples of suitabletranscriptional regulators include, e.g., Examples of transcriptionalregulators include, e.g., ABT1, ACYP2, AEBP1, AEBP2, AES, AFFI, AFF3,AHR, ANK1, ANK2, ANKFYl, ANKIBI, ANKRD1, ANKRD10, ANKRD2, ANKRD32,ANKRD46, ANKRD49, ANKRD56, ANKRD57, ANKS4B, AR, ARHGAP17, ARID1A,ARIDIB, ARID3A, ARID4A, ARIDSB, ARNT, ARNT2, ARNTL, ARNTL2, ARX, ASB10,ASB11, ASB12, ASB15, ASB2, ASBS, ASB8, ASB9, ASH1L, ASH2L, ASXL1, ASZ1,ATF1, ATF3, ATF4, ATF4, ATFS, ATF6, ATF7, ATF7IP, ATM, ATOH1, ATXN3,1300003B13RIK, B3GAT3, B930041F14RIK, BACH1, BACH2, BARX1, BARX2, BATF,BATF2, BATF3, BAZ2A, BBX, BC003267, BCL11A, BCL11B, BCL3, BCL6, BCL6B,BCLAF1, BCOR, BHLHA15, BHLHE40, BHLHE41, BLZF1, BMYC, BNC1, BNC2, BPNT1,BRCA1, BRWD1, BTBD11, BTF3, 6030408C04RIK, CAMK4, CARHSP1, CARM1, CBX4,CBX7, CCNC, CCNH, CCNT1, CCNT2, CDCSL, CDK2, CDK4, CDK9, CDKN2C, CDX1,CDX1, CDX2, CEBPA, CEBPB, CEBPD, CEBPG, CEBPG, CEBPZ, CHD4, CHD7, CHGB,CIC, CIITA, CITEDI, CITED2, CITED4, CLOCK, CLPB, CML3, CNOT7, COPS2,CREB1, CREB3, CREB3L1, CREB3L1, CREB3L2, CREB3L3, CREB5, CREBBP, CREBL2,CREM, CSDA, CSDA, CSDC2, CSDE1, CTBP2, CTCF, CTCFL, CTNNB1, CTNNBL1,CXXC1, D11BWG0517E, 2300002D11RIK, DACH1, DAXX, DBP, DDIT3, DDX20,DDX54, DDX58, DEAFI, DEK, DIDO1, DLX2, DMRT1, DMRT2, DMRTB1, DNMT1,DNMT3A, DR1, DRG1, DUSP26, DYSFIPI, E2F1, E2F2, E2F3, E2F5, E2F6, EBF1,EBF2, EBF3, EBF3, EED, EGRI, EGR2, EGR3, EHF, EHMT2, EID2, ELAVL2, ELF1,ELFI, ELF2, ELF3, ELF4, ELF5, ELK3, ELK4, ELL2, EMX2, EMX2, EN2, ENPP2,EOMES, EP300, EPAS1, ERF, ERG, ESR1, ESRRA, ESRRB, ESRRG, ETS1, ETS2,ETV1, ETV3, ETV4, ETV5, ETV6, EVIl, EWSR1, EZH1, EZH2, FAH, FBXL10,FBXL11, FBXW7, FEM1A, FEM1B, FEM1C, FHL2, FLII, FMNL2, FOS, FOSB, FOSL1,FOSL2, FOXA1, FOXA2, FOXA3, FOXCI, FOXD1, FOXD2, FOXD3, FOXF1, FOXF1A,FOXF2, FOXG1, FOXII, FOXJ2, FOXJ3, FOXK1, FOXK2, FOXL1, FOXL2, FOXM1,FOXN1, FOXN2, FOXN3, FOXO1, FOXO3, FOXPI, FOXP2, FOXP3, FOXP4, FOXQ1,FUS, FUSIP, 2810021G02RIK, GABPA, GABPB1, GARNL1, GAS7, GATA1, GATA2,GATA3, GATA4, GATA5, GATA5, GATA6, GBX2, GCDH, GCM1, GFI1, GFI1B, GLI2,GLI3, GLIS1, GLIS2, GLIS3, GLS2, GMEB1, GMEB2, GRHL1, GRHL2, GRHL3,GRLF1, GTF2A1, GTF2B, GTF2E2, GTF2F1, GTF2F2, GTF2H2, GTF2H4, GTF2I,GTF2IRD1, GTF2IRD1, GZF1, HAND2, HBP1, HCLS1, HDAC10, HDAC11, HDAC2,HDAC5, HDAC9, HELZ, HES1, HES4, HES5, HES6, HEXIM1, HEY2, HEYL, HHEX,HHEX, HIC1, HIC2, HIF1A, HIF1AN, HIPK2, HIVEP1, HIVEP2, HIVEP2, HIVEP3,HLF, HLTF, HLX, HMBOX1, HMG20A, HMGA2, HMGB2, HMGB3, HNF1B, HNF4A,HNF4G, HOMEZ, HOXA10, HOXAll, HOXA13, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6,HOXA7, HOXA9, HOXB1, HOXB2, HOXB3, HOXB4, HOXB6, HOXB7, HOXB8, HOXB9,HOXC10, HOXC10, HOXC11, HOXC5, HOXC6, HOXC8, HOXC9, HOXD8, HOXD9, HR,HSBP1, HSF2BP, HTATIP2, HTATSF1, HUWE1, 5830417I10RIK, ID1, ID2, ID3,ID3, IFNAR2, IKBKB, IKBKG, IKZF1, IKZF2, IKZF3, IKZF4, IL31RA, ILF3,INGI, ING2, ING3, ING4, INSM1, INTS12, IQWD1, IRF1, IRFI, IRF2, IRF3,IRF4, IRF5, IRF6, IRF7, IRF8, IRF8, IRX1, IRX2, IRX3, IRX4, IRX5, ISL1,ISL2, ISX, ISX, IVNS1ABP, 2810021J22RIK, JARID1A, JARIDIB, JARIDIC,JARIDID, JDP2, JUN, JUNB, JUND, KLF1, KLF10, KLF11, KLF12, KLF13, KLF15,KLF16, KLF2, KLF3, KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF9, KRR1,6330416L07RIK, L3MBTL2, LASS2, LASS4, LASS6, LBA1, LBH, LBX1, LCOR,LDB1, LDB2, LEFI, LHX1, LHX2, LHX5, LIMD1, LIN28, LMO1, LMO4, LMX1A,LSM11, LSM4, LYL1, 9030612M13RIK, 1810007M14RIK, 3632451006RIK, MAF,MAFA, MAFB, MAFF, MAFG, MAFK, MAGEDI, MAP3K12, MAPK1, MAPK3, MAPK8,MAPK8IP1, MAX, MAZ, MBD2, MCM2, MCM4, MCM5, MCM6, MCM7, MECOM, MECP2,MED12, MED8, MEF2A, MEF2B, MEF2C, MEF2D, MEIS1, MEIS1, MEIS2, MEOX2,MESP2, MIDI, MITF, MKI67IP, MKL1, MLL1, MLL3, MLLT10, MLLT3, MLX, MLXIP,MLXIPL, MNT, MNX1, MPL, MSC, MSRB2, MSX2, MTA3, MTF1, MTF2, MTPN, MXD1,MXD4, MXIl, MYB, MYBBPlA, MYBL2, MYC, MYCBP, MYCL1, MYCN, MYEF2, MYF6,MYNN, MYOCD, MYOD1, MYOG, MYST3, MYST4, MYT1L, MZF1, NAB1, NAB2, NANOG,NARGI, NCOA1, NCOA2, NCOA3, NCOR1, NCOR2, NDN, NEURODI, NEUROD4,NEUROD6, NEUROGI, NEUROG2, NFAT5, NFATC1, NFATC2, NFATC2IP, NFATC3,NFATC3, NFATC4, NFE2, NFE2L1, NFE2L2, NFIA, NFIA, NFIB, NFIC, NFIL3,NFIX, NFKB1, NFKB2, NFKBIB, NFKBIE, NFKBIZ, NFX1, NFXL1, NFYA, NFYB,NHLH1, NKX2-2, NKX2-3, NKX2-5, NKX2-6, NKX6-2, NMI, NOTCHI, NOTCH2,NOTCH3, NOTCH4, NPAS1, NPAS2, NPAS3, NROB1, NROB2, NR1D1, NR1D2, NR1H3,NR1H4, NR1I2, NR1I3, NR2C1, NR2C2, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1,NR3C2, NR4A1, NR4A2, NR4A2, NR4A3, NR5A1, NR5A2, NRARP, NRIP1, NRIP2,NSBP1, NSD1, NUDT12, NULL, NUPR1, 1700065013RIK, OLIGI, OLIG2, OLIG2,ONECUTI, ONECUT2, ONECUT3, ORC2L, OSGINI, OSR1, OSR2, OSTF1, OVOL1,OVOL2, PAPOLA, PAPOLG, PAPPA2, PATZ1, PAWR, PAX2, PAX5, PAX6, PAX7,PAX8, PAX9, PBX1, PBX2, PBX3, PBX4, PCBD1, PCGF6, PDCD11, PDLIM4, PDX1,PEG3, PERI, PFDN1, PGR, PHF1, PHF10, PHF12, PHF13, PHF14, PHF20, PHF21A,PHF5A, PHF7, PHOX2A, PHOX2B, PIAS2, PIR, PITX1, PITX2, PKNOX1, PKNOX2,PLA2G6, PLAGLI, PLAGL2, PLRG1, PML, POGK, POLR2B, POLR2E, POLR2H,POLR3E, POLR3H, POLRMT, POU1F1, POU2AF1, POU2F1, POU2F2, POU3F2, POU3F3,POU3F3, POU5F1, POU6F1, PPARA, PPARD, PPARG, PPARGC1A, PPARGClB,PPP1R12C, PPP1R13B, PPP1R16B, PPP1R1B, PPP2R1A, PPP3CB, PQBP1, PRDM1,PRDM14, PRDM15, PRDM16, PRDM2, PRDM4, PRDM5, PRDM6, PRDM8, PREB,PRKAR1A, PRKCBP1, PROX1, PRRX1, PRRX2, PSMC5, PSMD10, PSMD9, PTF1A,PTGES2, PURB, PWP1, RAB11A, RAB11B, RAB15, RAB18, RAB1B, RAB25, RAB8A,RAB8B, RAIl4, RARA, RARB, RARG, RASSF7, RB1, RBBP7, RBL1, RBM14, RBM39,RBM9, RBPJ, RBPJL, RCOR2, REL, RELA, RELB, RERE, REST, REXO4, RFC1,RFX1, RFX2, RFX3, RFX5, RFX7, RFX8, RHOX5, RHOX6, RHOX9, RIPK4, RNF12,RNF14, RNF141, RNF38, RNF4, RORA, RORA, RORB, RORC, RPS6KA4, RREB1,RSRC1, RUNX1, RUNX1T1, RUNX2, RUNX2, RUNX3, RUVBL1, RUVBL2, RXRA, RXRG,RYBP, SAFB2, SALL1, SALL1, SALL2, SALL4, SAP30, SAP30BP, SATB1, SATB2,SATB2, SCANDI, SCAP, SCRT2, SEC14L2, SERTADI, SF1, SFPI1, SFRS5, SH3D19,SH3PXD2B, SHANK3, SHOX2, SHPRH, SIN3A, SIN3B, SIRT2, SIRT3, SIRT5, SIX1,SIX1, SIX2, SIX3, SIX4, SIX5, SKI, SMAD1, SMAD2, SMAD3, SMAD7, SMARCA1,SMARCA2, SMARCA5, SMARCB1, SMYD1, SNAIl, SNAI2, SNAPC2, SNAPC4, SNIP1,SOLH, SOX1, SOX10, SOX11, SOX12, SOX13, SOX15, SOX17, SOX18, SOX2,SOX21, SOX4, SOX5, SOX6, SOX7, SOX8, SOX9, SP1, SP110, SP140L, SP2, SP3,SP4, SP6, SP8, SPDEF, SPEN, SPIl, SPIB, SQSTM1, SREBF1, SREBF2, SREBF2,SRF, SSBP2, SSBP3, SSBP4, SSRP1, ST18, STAGI, STAT1, STAT1, STAT2,STAT3, STAT4, STAT5A, STAT5B, STAT5B, STAT6, SUB1, SUZ12, TADA2L, TAF13,TAF5, TAF5L, TAF7, TAF9, TALl, TALl, TARDBP, TBPL1, TBR1, TBX1, TBX10,TBX15, TBX18, TBX2, TBX2, TBX20, TBX21, TBX3, TBX4, TBX5, TBX6, TCEA1,TCEA3, TCEAL1, TCEB3, TCERG1, TCF12, TCF15, TCF19, TCF20, TCF21, TCF21,TCF3, TCF4, TCF7, TCF7L2, TCFAP2A, TCFAP2B, TCFAP2C, TCFCP2L1, TCFE2A,TCFE3, TCFEB, TCFEC, TCFL5, TEAD1, TEAD2, TEAD3, TEAD4, TEF, TFAP2A,TFAP2C, TFCP2L1, TFDP2, TFEB, TFEC, TGFB1I1, TGIF1, TGIF2, TGIF2LX,THRA, THRAP3, THRB, THRSP, TIAL1, TLE1, TLE6, TMEM131, TMPO, TNFAIP3,TOB1, TOX4, TP63, TRERFI, TRIB3, TRIM24, TRIM28, TRIM30, TRIP13, TRIP4,TRIP6, TRP53, TRP53BP1, TRP63, TRPS1, TRPS1, TSC22D1, TSC22D2, TSC22D3,TSC22D4, TSHZ1, TSHZ1, TSHZ3, TTRAP, TUB, TULP4, TWISTI, TWIST2, TYSND1,UBE2W, UBN1, UBP1, UBTF, UGP2, UHRF1, UHRF2, UNCX, USF1, USF2, UTFI,VDR, VEZF1, VGLL2, VSX1, WASL, WHSC1, WHSC2, WT1, WWP1, WWTR1, XBP1,YAF2, YY1, ZBED1, ZBED4, ZBTB1, ZBTB10, ZBTB16, ZBTB16, ZBTB17, ZBTB2,ZBTB20, ZBTB22, ZBTB25, ZBTB32, ZBTB38, ZBTB4, ZBTB43, ZBTB45, ZBTB47,ZBTB7A, ZBTB7B, ZBTB7C, ZCCHC8, ZDHHC13, ZDHHC16, ZDHHC21, ZDHHC5,ZDHHC6, ZEB2, ANK2ZEB2, ZFHX2, ZFHX3, ZFHX4, ZFP105, ZFP110, ZFP143,ZFP148, ZFP161, ZFP192, ZFP207, ZFP219, ZFP238, ZFP263, ZFP275, ZFP277,ZFP281, ZFP287, ZFP292, ZFP35, ZFP354C, ZFP36, ZFP36L1, ZFP386, ZFP407,ZFP42, ZFP423, ZFP426, ZFP445, ZFP451, ATF5ZFP451, ZFP467, ZFP52, ZFP57,ZFP592, ZFP593, ZFP597, ZFP612, ZFP637, ZFP64, ZFP647, ZFP748, ZFP810,ZFP9, ZFP91, ZFPM1, ZFPM2, ZFX, ZHX2, ZHX3, ZICI, ZIC2, ZIC3, ZIC4,ZICS, ZKSCAN1, ZKSCAN3, ZMYND11, ZNF143, ZNF160, ZNF175, ZNF184, ZNF192,ZNF213, ZNF217, ZNF219, ZNF22, ZNF238, ZNF24, ZNF267, ZNF273, ZNF276,ZNF280D, ZNF281, ZNF292, ZNF311, ZNF331, ZNF335, ZNF337, ZNF33B, ZNF366,ZNF394, ZNF398, ZNF41, ZNF410, ZNF415, ZNF423, ZNF436, ZNF444, ZNF445,ZNF451, ZNF460, ZNF496, ZNF498, ZNF516, ZNF521, ZNF532, ZNF536, ZNF546,ZNF552, ZNF563, ZNF576, ZNF580, ZNF596, ZNF621, ZNF628, ZNF648, ZNF649,ZNF652, ZNF655, ZNF664, ZNF668, ZNF687, ZNF692, ZNF696, ZNF697, ZNF710,ZNF80, ZNF91, ZNF92, ZNRD1, ZSCAN10, ZSCAN16, ZSCAN20, ZSCAN21, ZXDC,andZZZ3.

In some cases, the intracellular domain is a transcription factor.Suitable transcription factors include, e.g., ASCL1, BRN2, CDX2, CDX4,CTNNB1, EOMES, JUN, FOS, HNF4a, HOXAs (e.g., HOXA1, HOXA2, HOXA3, HOXA4,HOXAS, HOXA10, HOXAll, HOXA13), HOXBs (e.g., HOXB9), HOXCs (e.g., HOXC4,HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXCl1, HOXC12, HOXC13), HOXDs(e.g., HOXD1, HOXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOXD11, HOXD12,HOXD13), SNAIl-3, MYOD1, MYOG, NEUROD1-6 (e.g., NEUODI, NEUROD2,NEUROD4, NEUROD6), PDX1, PU.1, SOX2, Nanog, Kf4, BCL-6, SOX9, STAT1-6,TBET, TCF, TEAD1-4 (e.g., TEADI, TEAD2, TEAD3, TEAD4), TAF6L, CLOCK,CREB, GATA3, IRF7, MycC, NFkB, RORyt, RUNX1, SRF, TBX21, NFAT, MEF2D,and FoxP3.

In some cases, the intracellular domain is a transcription factor havinga regulatory role in one or more immune cells (i.e., an immune cellregulatory transcription factor). Suitable immune cell regulatorytranscription factors include, e.g., 2210012G02Rik, Akap81, App2,Arid4b, Arid5b, Ash1, Atf7, Atm, C430014K11Rik, Chd9, Dmtf1, Fos, Foxo1,Foxp1, Hmboxl, Kdm5b, Klf2, Mga, Mll1, Ml3, Myst4, Pcgf6, Rev31, Scml4,Scp2, Smarca2, Ssbp2, Suhw4, Tcf7, Tfdp2, Tox, Zbtb20, Zbtb44, Zeb1,Zfm1, Zfp1, Zfp319, Zfp329, Zfp35, Zfp386, Zfp445, Zfp518, Zfp652,Zfp827, Zhx2, Eomes, Arntl, Bbx, Hbpl, Jun, Mef2d, Mterfdl, Nfat5,Nfe212, Nrld2, Phf21a, Taf4b, Trf, Zbtb25, Zfp326, Zfp451, Zfp58,Zfp672, Egr2, Ikzf2, TafId, Chrac1, Dnajb6, Aplp2, Batf, Bhlhe40, Fosb,Hist1h1c, Hopx, Ifih1, Ikzf3, Lass4, Lin54, Mxd1, Mxi1, Prdm1, Prf1,Rora, Rpa2, Sap30, Stat2, Stat3, Taf9b, Tbx21, Trps1, Xbpl, Zeb2, Atf3,Cenpcl, Lass6, Rb1, Zbtb41, Crem, Fos12, Gtf2b, Irf7, Maff, Nr4a1,Nr4a2, Nr4a3, Obfc2a, Rbl2, Rel, Rybp, Sral, Tgifl, Tnfaip3, Uhrf2,Zbtbl, Ccdc124, Csda, E2f3, Epas1, Hf, H2afz, Hif1a, Ikzf5, Irf4, Nsbp1,Pim1, Rfc2, Swap70, Tfbm, 2610036L11Rik, 5133400G04Rik, Apitd1, Blm,Brca1, Brip1, Cld, C79407, Cenpa, Cfl1, Clspn, Ddx1, Dscc1, E2, E2f8,Ercc61, Ezh2, Fen1, Foxm1, Gen1, Gsg2, H2afx, Hdac1, Hdgf, Hells,Histhle, Hist3h2a, Hjurp, Hmgb2, Hmgb3, Irfl, Irf8, Kif22, Kif4, Lig,Lmo2, Lnp, Mbd4, Mcm2, Mcm3, Mcm4, Mcm5, Mcm6, Mcm7, Mybl2, Neil3,Nusap1, Orc61, Pola1, Pola2, Pole, Pole2, Polh, Polr2f, Polr2j, Ppplr8,Prim2, Psmc3ip, Rad51, Rad51c, Rad541, Rfc3, Rfc4, Rnps1, Rpa1, Smarcc1,Spic, Ssrp1, Taf9, Tfdp1, Tmpo, Topbp1, Trdmt1, Uhrf1, Wdhd1, Whsc1,Zbp1, Zbtb32, Zfp367, Carl, Polg2, Atr, Lef1, Myc, Nucb2, Satbi, Tafla,Ift57, Apex, Chd7, Chtf8, Ctnnbl, Etv3, Irf9, Myb, Mybbpla, Pms2, Preb,Sp110, Stat1, Trp53, Zfp414, App, Cdk9, Ddbl, Hsf2, Lbr, Pa2g4, Rbms,Rfcl, Rfc5, Tada21, Tex261, Xrcc6, and the like.

In some cases, a transcription factor may be an artificial transcriptionfactor (ATF) including but not limited to e.g., Zinc-finger-basedartificial transcription factors (including e.g., those described inSera T. Adv Drug Deliv Rev. 2009 61(7-8):513-26; Collins et al. CurrOpin Biotechnol. 2003 14(4):371-8; Onori et al. BMC Mol Biol. 2013 14:3the disclosures of which are incorporated herein by reference in theirentirety).

For example, in some cases, the intracellular domain comprises an aminoacid sequence having at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the Apoptosis-antagonizing transcription factor(AATF) amino acid sequence depicted in FIG. 37.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the Activator of basal transcription (ABT1) aminoacid sequence depicted in FIG. 38.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the adipocyte enhancer binding protein 2 amino acidsequence depicted in FIG. 39.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the activating transcription factor 1 (ATFI) aminoacid sequence depicted in FIG. 40.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the transcription regulator protein BACH1 aminoacid sequence depicted in FIG. 41.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the class E basic helix-loop-helix protein 41 aminoacid sequence depicted in FIG. 42.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the bromodomain-containing protein amino acidsequence depicted in FIG. 43.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the CCAAT/enhancer-binding protein zeta amino acidsequence depicted in FIG. 44.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the chromodomain-helicase-DNA-binding protein 1amino acid sequence depicted in FIG. 45.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the death-inducer obliterator 1 isoform c aminoacid sequence depicted in FIG. 46.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the protein Dr1 amino acid sequence depicted inFIG. 47.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the early growth response protein 1 amino acidsequence depicted in FIG. 48.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the ETS-related transcription factor Elf-2 aminoacid sequence depicted in FIG. 49.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the estrogen receptor amino acid sequence depictedin FIG. 50.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the zinc finger and BTB domain-containing protein7A amino acid sequence depicted in FIG. 51.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the four and a half LIM domains protein 1 aminoacid sequence depicted in FIG. 52.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the forkhead box protein P3 amino acid sequencedepicted in FIG. 53.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the GA-binding protein alpha chain amino acidsequence depicted in FIG. 54.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the hepatic leukemia factor amino acid sequencedepicted in FIG. 55.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the HOP amino acid sequence depicted in FIG. 56.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the DNA-binding protein inhibitor ID-1 amino acidsequence depicted in FIG. 57.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the DNA-binding protein inhibitor ID-2 (dominantnegative helix-loop-helix) amino acid sequence depicted in FIG. 58.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the interferon regulatory factor 1 amino acidsequence depicted in FIG. 59.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the Krueppel-like factor 12 amino acid sequencedepicted in FIG. 60.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the LIM domain-binding protein 1 amino acidsequence depicted in FIG. 61.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the LIM/homeobox protein Lhx1 amino acid sequencedepicted in FIG. 62.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the zinc finger transcription factor E2S-VP64 aminoacid sequence depicted in FIG. 63. As another example, in some cases,the intracellular domain comprises an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:VDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSGGSGGSGGSLEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK (SEQ ID NO:67);and has a length of 105-115 amino acids (e.g., 105, 106, 107, 108, 109,110, 111, 112, 113, 114, or 115 amino acids).

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the GAL4 DNA binding domain amino acid sequencedepicted in FIG. 64. As another example, in some cases, theintracellular domain comprises an amino acid sequence having at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGKGGSGGSGGSMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAA (SEQ ID NO:68); and has a length of from200 amino acids to 210 amino acids (e.g., 200, 201, 202, 203, 204, 205,206, 207, 208, 209, or 210 amino acids).

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the signal transducer and activator oftranscription 3 (STAT3) amino acid sequence depicted in FIG. 65.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the Myc amino acid sequence depicted in FIG. 66.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the ASCL1 amino acid sequence depicted in FIG. 67.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the CDX2 amino acid sequence depicted in FIG. 68.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the CREB1 amino acid sequence depicted in FIG. 69.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the CTNNB1 amino acid sequence depicted in FIG. 70.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the EOMES amino acid sequence depicted in FIG. 71.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the Fos amino acid sequence depicted in FIG. 72.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the GATA3 amino acid sequence depicted in FIG. 73.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the HOXA1 amino acid sequence depicted in FIG. 74.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the interferon regulatory factor 7 (IRF7) aminoacid sequence depicted in FIG. 75.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the Jun amino acid sequence depicted in FIG. 76.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the myocyte enhancer factor 2D (MEF2D) amino acidsequence depicted in FIG. 77.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the neuronal differentiation factor 1 (NEURODI)amino acid sequence depicted in FIG. 78.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the NFAT amino acid sequence depicted in FIG. 79.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the NFKB amino acid sequence depicted in FIG. 80.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the SNAIl amino acid sequence depicted in FIG. 81.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the STAT1 amino acid sequence depicted in FIG. 82.

As another example, in some cases, the intracellular domain comprises anamino acid sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the TEAD1 amino acid sequence depicted in FIG. 83.

In some embodiments, the intracellular domain is a transcriptionalactivator. In some cases, the intracellular domain comprises an aminoacid sequence having at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the following tetracycline-controlledtranscriptional activator (tTA) amino acid sequence:MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG (SEQ ID NO:69); and has a length of from about 245 aminoacids to 252 amino acids (e.g., 248, 249, 250, 251, or 252 amino acids).

In some embodiments, the intracellular domain is a transcriptionalactivator. In some cases, the transcriptional activator is GAL4-VP16. Insome cases, the transcriptional activator is GAL4-VP64. In some cases,the transcriptional activator is Tbx21. In some cases thetranscriptional activator is an engineered protein, such as a zincfinger or TALE based DNA binding domain fused to an effector domain suchas VP64 (transcriptional activation) or KRAB (transcriptionalrepression). A variety of other transcriptional transactivators areknown in the art is suitable for use.

In some cases, the intracellular domain comprises an amino acid sequencehaving at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the following GAL4-VP64 sequence:MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS (SEQ ID NO:70); and has a length offrom 208 to 214 amino acids (e.g., 208, 209, 210, 211, 212, 213, or 214amino acids).

In some cases, the intracellular domain comprises an amino acid sequencehaving at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the following Tbx21 sequence:MGIVEPGCGDMLTGTEPMPGSDEGRAPGADPQHRYFYPEPGAQDADERRGGGSLGSPYPGGALVPAPPSRFLGAYAYPPRPQAAGFPGAGESFPPPADAEGYQPGEGYAAPDPRAGLYPGPREDYALPAGLEVSGKLRVALNNHLLWSKFNQHQTEMIITKQGRRMFPFLSFTVAGLEPTSHYRMFVDVVLVDQHHWRYQSGKWVQCGKAEGSMPGNRLYVHPDSPNTGAHWMRQEVSFGKLKLTNNKGASNNVTQMIVLQSLHKYQPRLHIVEVNDGEPEAACNASNTHIFTFQETQFIAVTAYQNAEITQLKIDNNPFAKGFRENFESMYTSVDTSIPSPPGPNCQFLGGDHYSPLLPNQYPVPSRFYPDLPGQAKDVVPQAYWLGAPRDHSYEAEFRAVSMKPAFLPSAPGPTMSYYRGQEVLAPGAGWPVAPQYPPKMGPASWFRPMRTLPMEPGPGGSEGRGPEDQGPPLVWTEIAPIRPESSDSGLGEGDSKRRRVSPYPSSGDSSSPAGAPSPFDKEAEG QFYNYFPN(SEQ ID NO:71); and has a length of from 530 amino acids to 540 aminoacids (e.g., 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, or 540amino acids).

In some cases, the intracellular domain comprises an amino acid sequencehaving at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the following MyoD amino acid sequence:MELLSPPLRDIDLTGPDGSLCSFETADDFYDDPCFDSPDLRFFEDLDPRLVHMGALLKPEEHAHFPTAVHPGPGAREDEHVRAPSGHHQAGRCLLWACKACKRKTTNADRRKAATMRERRRLSKVNEAFETLKRCTSSNPNQRLPKVEILRNAIRYIEGLQALLRDQDAAPPGAAAFYAPGPLPPGRGSEHYSGDSDASSPRSNCSDGMMDYSGPPSGPRRQNGYDTAYYSEAARESRPGKSAAVSSLDCLSSIVERISTDSPAAPALLLADAPPESPPGPPEGASLSDTEQGTQTPSPDAAPQCPAGSNPNAIYQVL (SEQ ID NO:72); and has a length of from 305 to325 amino acids (e.g., 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, or 325 amino acids).

In some cases, the intracellular domain comprises a toxin. Examples oftoxins include, e.g., diphtheria toxin A fragment, nonbinding activefragments of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa),ricin A chain, abrin A chain, modeccin A chain, α-sacrin, certainAleurites fordii proteins, certain Dianthin proteins, Phytolaccaamericana proteins (PAP, PAPII and PAP-S), Morodica charantia inhibitor,curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogillin,restrictocin, phenomycin, and neomycin. In some cases, the intracellulardomain comprises a protein that is normally secreted by a bacterialpathogen via a Type II secretion system. In some cases, theintracellular domain comprises a toxic bacterial effector from Type III(e.g., Salmonella, Shigella, Yersinia, Vibrio) and type IV (e.g.,Bordetella pertussis, Legionella pneumophila, Agrobacterium tumefaciens)secretion systems. Examples of toxic bacterial effectors from Type IIIbacterial secretion systems include, e.g., VopQ, YopH, and the like.See, e.g., Dean (2011) FEMS Microbiol. Rev. 35:1100. Examples of toxicbacterial effectors from Type IV bacterial secretion systems include,e.g., pertussis toxin, CagA, and the like.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure is a hormone. Examples of suitablehormones include, e.g., erythropoietin (EPO), insulin, secretins,glucagon-like polypeptide 1 (GLP-1), and the like. Further examples ofsuch hormones include, but are not limited to, activin, inhibin,adiponectin, adipose-derived hormones, adrenocorticotropic hormone,Afamelanotide, agouti signaling peptide, Allatostatin, Amylin, Amylinfamily, angiotensin, atrial natriuretic peptide, gastrin, somatotropin,bradykinin, brain-derived neurotrophic factor, calcitonin,cholecystokinin, ciliary neurotrophic factor, corticotropin-releasinghormone, cosyntropin, endothelian, enteroglucagon, fibroblast growthfactor 15 (FGF15), GFG15/19, follicle-stimulating hormone, gastrin,gastroinhibitory peptide, ghrelin, glucagon, glucagon-like peptide-1,gonadotropin, gonadotropin-releasing hormone,granulocyte-colony-stimulating factor, growth hormone,growth-hormone-releasing hormone, hepcidin, human chorionicgonadotropin, human placental lactogen, incretin, insulin, insulinanalog, insulin aspart, insulin degludec, insulin glargine, insulinlispro, insulin-like growth factor, insulin-like growth factor-1,insulin-like growth factor-2, leptin, liraglutide, luteinizing hormone,melanocortin, melanocyte-stimulating hormone,alpha-melanocyte-stimulating hormone, melanotin II, minigastrin,N-terminal prohormone of brain natriuretic peptide, nerve growth factor,neurotrophin-3, neurotrophin-4, NPH insulin, obestatin, orexin,osteocalcin, pancreatic hormone, parathyroid hormone, peptide hormone,peptide YY, plasma renin activity, pramlintide, preprohormone,prolactin, relaxin, relaxin family peptide hormone, renin, salcatonin,secretin, secretin family peptide hormone, sincalide, teleost leptins,temporin, tesamorelin, thyroid-stimulating hormone,thyrotropin-releasing hormone, urocortin, urocortin II, urocortin III,vasoactive intestinal peptide, and vitellogenin.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure is a growth factor. Examples ofsuitable growth factors include, but are not limited to, hepatocytestimulating factor, plasmacytoma growth factor, brain derivedneurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF),neurotrophic factor 3 (NT3), fibroblast growth factor (FGF),transforming growth factor (TGF), platelet transforming growth factor,milk growth factor, endothelial growth factors (EGF), endothelialcell-derived growth factors (ECDGF), alpha-endothelial growth factor,beta-endothelial growth factor, neurotrophic growth factor, nerve growthfactor (NGF), vascular endothelial growth factor (VEGF), 4-1 BB receptor(4-1BBR), TRAIL (TNF-related apoptosis inducing ligand), artemin(GFRalpha3-RET ligand), BCA-1 (B cell-attracting chemokinel), Blymphocyte chemoattractant (BLC), B cell maturation protein (BCMA),brain-derived neurotrophic factor (BDNF), bone growth factor such asosteoprotegerin (OPG), bone-derived growth factor, megakaryocyte derivedgrowth factor (MGDF), keratinocyte growth factor (KGF), thrombopoietin,platelet-derived growth factor (PGDF), megakaryocyte derived growthfactor (MGDF), keratinocyte growth factor (KGF), platelet-derived growthfactor (PGDF), neurotrophin-2 (NT-2), neurotrophin-3 (NT-3),neurotrophin-4 (NT4), neurotrophin-5 (NT-5), glial cell line-derivedneurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), boneMorphogenetic protein 2 (BMP2), granulocyte macrophage colonystimulating factor (GM-CSF), granulocyte colony stimulating factor(G-CSF), macrophage colony stimulating factor (M-CSF), colonystimulating factor (CSF), and the like.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure is a cytokine. Examples ofsuitable cytokines include, e.g., interferons (e.g., analpha-interferon, a beta-interferon, a gamma-interferon); interleukins(e.g., IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10 IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-17A, IL-18,IL-19, IL-20, IL-24); tumor necrosis factors (e.g., TNF-α); transforminggrowth factor-beta; TRAIL; and the like. Examples of suitable cytokinesalso include flexi-12 (Anderson et al. (1997) Hum. Gene Ther. 8:1125), asingle chain polypeptide that combines the two polypeptide chains of anIL-12 heterodimer); IL-12 superkine H9 (Levin et al. (2012) Nature484:529); and the like.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure is a chemokine. Examples ofsuitable chemokines include, e.g., MIP-1, MIP-10, MCP-1, RANTES, IP10,and the like. Additional examples of suitable chemokines include, butare not limited to, chemokine (C-C motif) ligand-2 (CCL2; also referredto as monocyte chemotactic protein-1 or MCP1); chemokine (C-C motif)ligand-3 (CCL3; also known as macrophage inflammatory protein-iA orMIP1A); chemokine (C-C motif) ligand-5 (CCL5; also known as RANTES);chemokine (C-C motif) ligand-17 (CCL17; also known as thymus andactivation regulated chemokine or TARC); chemokine (C-C motif) ligand-19(CCL19; also known as EBI1 ligand chemokine or ELC); chemokine (C-Cmotif) ligand-21 (CCL21; also known as 6Ckine); C-C chemokine receptortype 7 (CCR7); chemokine (C-X-C motif) ligand 9 (CXCL9; also known asmonokine induced by gamma interferon or MIG); chemokine (C-X-C motif)ligand 10 (CXCL10; also known as interferon gamma-induced protein 10 orIP-10); chemokine (C-X-C motif) ligand 11 (CXCL11; also calledinterferon-inducible T-cell alpha chemoattractant or I-TAC); chemokine(C-X-C motif) ligand 16 (CXCL16; chemokine (C motif) ligand (XCL1; alsoknown as lymphotactin); and macrophage colony-stimulating factor (MCSF).

In some cases, the intracellular domain of a binding triggeredtranscriptional switch, e.g., a chimeric Notch receptor polypeptide, ofthe present disclosure is an antibody (or an antigen-binding fragment ofan antibody). Suitable antibodies include, e.g., Natalizumab (Tysabri;Biogen Idec/Elan) targeting a4 subunit of a401 anda407 integrins (asused in the treatment of MS and Crohn's disease); Vedolizumab (MLN2;Millennium Pharmaceuticals/Takeda) targeting a407 integrin (as used inthe treatment of UC and Crohn's disease); Belmumab (Benlysta; HumanGenome Sciences/GlaxoSmithKline) targeting BAFF (as used in thetreatment of SLE); Atacicept (TACI-Ig; Merck/Serono) targeting BAFF andAPRIL (as used in the treatment of SLE); Alefacept (Amevive; Astellas)targeting CD2 (as used in the treatment of Plaque psoriasis, GVHD);Otelixizumab (TRX4; Tolerx/GlaxoSmithKline) targeting CD3 (as used inthe treatment of TID); Teplizumab (MGA031; MacroGenics/Eli Lilly)targeting CD3 (as used in the treatment of T1D); Rituximab(Rituxan/Mabthera; Genentech/Roche/Biogen Idec) targeting CD20 (as usedin the treatment of Non-Hodgkin's lymphoma, RA (in patients withinadequate responses to TNF blockade) and CLL); Ofatumumab (Arzerra;Genmab/GlaxoSmithKline) targeting CD20 (as used in the treatment of CLL,RA); Ocrelizumab (2H7; Genentech/Roche/Biogen Idec) targeting CD20 (asused in the treatment of RA and SLE); Epratuzumab (hLL2;Immunomedics/UCB) targeting CD22 (as used in the treatment of SLE andnon-Hodgkin's lymphoma); Alemtuzumab (Campath/MabCampath; Genzyme/Bayer)targeting CD52 (as used in the treatment of CLL, MS); Abatacept(Orencia; Bristol-Myers Squibb) targeting CD80 and CD86 (as used in thetreatment of RA and JIA, UC and Crohn's disease, SLE); Eculizumab(Soliris; Alexion pharmaceuticals) targeting C5 complement protein (asused in the treatment of Paroxysmal nocturnal haemoglobinuria);Omalizumab (Xolair; Genentech/Roche/Novartis) targeting IgE (as used inthe treatment of Moderate to severe persistent allergic asthma);Canakinumab (Ilaris; Novartis) targeting IL-1β (as used in the treatmentof Cryopyrin-associated periodic syndromes, Systemic JIA, neonatal-onsetmultisystem inflammatory disease and acute gout); Mepolizumab (Bosatria;GlaxoSmithKline) targeting IL-5 (as used in the treatment ofHyper-eosinophilic syndrome); Reslizumab (SCH55700; CeptionTherapeutics) targeting IL-5 (as used in the treatment of Eosinophilicoesophagitis); Tocilizumab (Actemra/RoActemra; Chugai/Roche) targetingIL-6R (as used in the treatment of RA, JIA); Ustekinumab (Stelara;Centocor) targeting IL-12 and IL-23 (as used in the treatment of Plaquepsoriasis, Psoriatic arthritis, Crohn's disease); Briakinumab (ABT-874;Abbott) targeting IL-12 and IL-23 (as used in the treatment of Psoriasisand plaque psoriasis); Etanercept (Enbrel; Amgen/Pfizer) targeting TNF(as used in the treatment of RA, JIA, psoriatic arthritis, AS and plaquepsoriasis); Infliximab (Remicade; Centocor/Merck) targeting TNF (as usedin the treatment of Crohn's disease, RA, psoriatic arthritis, UC, AS andplaque psoriasis); Adalimumab (Humira/Trudexa; Abbott) targeting TNF (asused in the treatment of RA, JIA, psoriatic arthritis, Crohn's disease,AS and plaque psoriasis); Certolizumab pegol (Cimzia; UCB) targeting TNF(as used in the treatment of Crohn's disease and RA); Golimumab(Simponi; Centocor) targeting TNF (as used in the treatment of RA,psoriatic arthritis and AS); and the like. In some cases, the antibodywhose production is induced by the intracellular domain of a synNotchpolypeptide of the present disclosure is a therapeutic antibody for thetreatment of cancer. Such antibodies include, e.g., Ipilimumab targetingCTLA-4 (as used in the treatment of Melanoma, Prostate Cancer, RCC);Tremelimumab targeting CTLA-4 (as used in the treatment of CRC, Gastric,Melanoma, NSCLC); Nivolumab targeting PD-1 (as used in the treatment ofMelanoma, NSCLC, RCC); MK-3475 targeting PD-1 (as used in the treatmentof Melanoma); Pidilizumab targeting PD-1 (as used in the treatment ofHematologic Malignancies); BMS-936559 targeting PD-Li (as used in thetreatment of Melanoma, NSCLC, Ovarian, RCC); MED14736 targeting PD-L;MPDL33280A targeting PD-L (as used in the treatment of Melanoma);Rituximab targeting CD20 (as used in the treatment of Non-Hodgkin'slymphoma); Ibritumomab tiuxetan and tositumomab (as used in thetreatment of Lymphoma); Brentuximab vedotin targeting CD30 (as used inthe treatment of Hodgkin's lymphoma); Gemtuzumab ozogamicin targetingCD33 (as used in the treatment of Acute myelogenous leukaemia);Alemtuzumab targeting CD52 (as used in the treatment of Chroniclymphocytic leukaemia); IGN101 and adecatumumab targeting EpCAM (as usedin the treatment of Epithelial tumors (breast, colon and lung));Labetuzumab targeting CEA (as used in the treatment of Breast, colon andlung tumors); huA33 targeting gpA33 (as used in the treatment ofColorectal carcinoma); Pemtumomab and oregovomab targeting Mucins (asused in the treatment of Breast, colon, lung and ovarian tumors); CC49(minretumomab) targeting TAG-72 (as used in the treatment of Breast,colon and lung tumors); cG250 targeting CAIX (as used in the treatmentof Renal cell carcinoma); J591 targeting PSMA (as used in the treatmentof Prostate carcinoma); MOv18 and MORAb-003 (farletuzumab) targetingFolate-binding protein (as used in the treatment of Ovarian tumors);3F8, chi4.18 and KW-2871 targeting Gangliosides (such as GD2, GD3 andGM2) (as used in the treatment of Neuroectodermal tumors and someepithelial tumors); hu3S193 and IgN311 targeting Le y (as used in thetreatment of Breast, colon, lung and prostate tumors); Bevacizumabtargeting VEGF (as used in the treatment of Tumor vasculature); IM-2C6and CDP791 targeting VEGFR (as used in the treatment ofEpithelium-derived solid tumors); Etaracizumab targeting Integrin_V_3(as used in the treatment of Tumor vasculature); Volociximab targetingIntegrin_5_1 (as used in the treatment of Tumor vasculature); Cetuximab,panitumumab, nimotuzumab and 806 targeting EGFR (as used in thetreatment of Glioma, lung, breast, colon, and head and neck tumors);Trastuzumab and pertuzumab targeting ERBB2 (as used in the treatment ofBreast, colon, lung, ovarian and prostate tumors); MM-121 targetingERBB3 (as used in the treatment of Breast, colon, lung, ovarian andprostate, tumors); AMG 102, METMAB and SCH 900105 targeting MET (as usedin the treatment of Breast, ovary and lung tumors); AVE1642, IMC-A12,MK-0646, R1507 and CP 751871 targeting IGF1R (as used in the treatmentof Glioma, lung, breast, head and neck, prostate and thyroid cancer);KB004 and IIIA4 targeting EPHA3 (as used in the treatment of Lung,kidney and colon tumors, melanoma, glioma and haematologicalmalignancies); Mapatumumab (HGS-ETR1) targeting TRAILR1 (as used in thetreatment of Colon, lung and pancreas tumors and haematologicalmalignancies); HGS-ETR2 and CS-1008 targeting TRAILR2; Denosumabtargeting RANKL (as used in the treatment of Prostate cancer and bonemetastases); Sibrotuzumab and F19 targeting FAP (as used in thetreatment of Colon, breast, lung, pancreas, and head and neck tumors);81C6 targeting Tenascin (as used in the treatment of Glioma, breast andprostate tumors); Blinatumomab (Blincyto; Amgen) targeting CD3 (as usedin the treatment of ALL); pembrolizumab targeting PD-1 as used in cancerimmunotherapy; 9E10 antibody targeting c-Myc; and the like.

Antibodies that may find use, in whole or in part, in the intracellulardomain of a binding triggered transcriptional switch also include butare not limited to 8H9, Abagovomab, Abciximab, Abituzumab, Abrilumab,Actoxumab, Aducanumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518,Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox,Anetumab ravtansine, Anifrolumab, Anrukinzumab, Apolizumab, Arcitumomab,Ascrinvacumab, Aselizumab, Atezolizumab, Atinumab,Atlizumab/tocilizumab, Atorolimumab, Bapineuzumab, Basiliximab,Bavituximab, Bectumomab, Begelomab, Benralizumab, Bertilimumab,Besilesomab, Bevacizumab/Ranibizumab, Bezlotoxumab, Biciromab,Bimagrumab, Bimekizumab, Bivatuzumab mertansine, Blosozumab,Bococizumab, Brentuximabvedotin, Brodalumab, Brolucizumab,Brontictuzumab, Cantuzumab mertansine, Cantuzumab ravtansine,Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab,cBR96-doxorubicin immunoconjugate, Cedelizumab, Ch.14.18, Citatuzumabbogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumabtetraxetan, Codrituzumab, Coltuximab ravtansine, Conatumumab,Concizumab, CR6261, Crenezumab, Dacetuzumab, Daclizumab, Dalotuzumab,Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumabmafodotin, Derlotuximab biotin, Detumomab, Dinutuximab, Diridavumab,Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Durvalumab,Dusigitumab, Ecromeximab, Edobacomab, Edrecolomab, Efalizumab,Efungumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab,Emibetuzumab, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol,Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomabcituxetan, Erlizumab, Ertumaxomab, Etrolizumab, Evinacumab, Evolocumab,Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05,Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Firivumab,Flanvotumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab,Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab,Gavilimomab, Gevokizumab, Girentuximab, Glembatumumab vedotin,Gomiliximab, Guselkumab, Ibalizumab, Ibalizumab, Icrucumab,Idarucizumab, Igovomab, IMAB362, Imalumab, Imciromab, Imgatuzumab,Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Inolimomab,Inotuzumab ozogamicin, Intetumumab, Iratumumab, Isatuximab, Itolizumab,Ixekizumab, Keliximab, Lambrolizumab, Lampalizumab, Lebrikizumab,Lemalesomab, Lenzilumab, Lerdelimumab, Lexatumumab, Libivirumab,Lifastuzumab vedotin, Ligelizumab, Lilotomab satetraxetan, Lintuzumab,Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine,Lucatumumab, Lulizumab pegol, Lumiliximab, Lumretuzumab, Margetuximab,Maslimomab, Matuzumab, Mavrilimumab, Metelimumab, Milatuzumab,Minretumomab, Mirvetuximab soravtansine, Mitumomab, Mogamulizumab,Morolimumab, Morolimumab immune, Motavizumab, Moxetumomab pasudotox,Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox,Narnatumab, Nebacumab, Necitumumab, Nemolizumab, Nerelimomab,Nesvacumab, Nofetumomab merpentan, Obiltoxaximab, Obinutuzumab,Ocaratuzumab, Odulimomab, Olaratumab, Olokizumab, Onartuzumab,Ontuxizumab, Opicinumab, Oportuzumab monatox, Orticumab, Otlertuzumab,Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Pankomab,Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab,Patritumab, Perakizumab, Pexelizumab, Pinatuzumab vedotin, Pintumomab,Placulumab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab,Pritumumab, PRO 140, Quilizumab, Racotumomab, Radretumab, Rafivirumab,Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab,Regavirumab, Rilotumumab, Rinucumab, Robatumumab, Roledumab,Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Sacituzumabgovitecan, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab,Seribantumab, Setoxaximab, Sevirumab, SGN-CD19A, SGN-CD33A, Sifalimumab,Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Sofituzumab vedotin,Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab,Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab,Tanezumab, Taplitumomab paptox, Tarextumab, Tefibazumab, Telimomabaritox, Tenatumomab, Teneliximab, Teprotumumab, Tesidolumab, Tetulomab,TGN1412, Ticilimumab/tremelimumab, Tigatuzumab, Tildrakizumab, TNX-650,Toralizumab, Tosatoxumab, Tovetumab, Tralokinumab, TRBS07, Tregalizumab,Trevogrumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab,Ulocuplumab, Urelumab, Urtoxazumab, Vandortuzumab vedotin, Vantictumab,Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Veltuzumab,Vepalimomab, Vesencumab, Visilizumab, Vorsetuzumab mafodotin, Votumumab,Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab, Zolimomab aritox, andthe like.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure is a neuropeptide. Examples ofsuitable neuropeptides include, but are not limited to,N-Acetylaspartylglutamic acid, agouti-related peptide, alpha-endorphin,big dynorphin, bombesin, bombesin-like peptides, carbetocin,cocaine-and-amphetamine regulated transcript (CART), cholecystokinin,corazonin, corticotropin-like intermediate peptide, cortistatin,demoxytocin, dynorphin A, dynorphin B, eledoisin, enkephalin, galanin,galanin-like peptide, galmic, galnon, gamma-endorphin, ghrelin,hemopressin, kisspeptin, neurokinin B, neuromedin B, neuromedin N,neuromedin S, neuromedin U, neuromedin S, neuromedin Y, neuropeptide Y,neurotensin, nociceptin, opiorphin, orexin, orexin-A, oxytocin,physalaemin, preprotachykinin, proctolin, proenkephalin,poopiomelanocortin, protein episteme, relaxin-3, somatostatin, substanceP, TAC1, tachykinin peptides, vasopressin, and vasotocin.

Gene Products Induced by a Released Intracellular Domain of a synNotchPolypeptide

In some cases, the intracellular domain is a polypeptide that, whenreleased upon binding of the first member of the specific binding pairto a second member of the specific binding pair, induces production, ina cell that expresses the chimeric Notch polypeptide, of a gene product.For example, in some cases, the intracellular domain of a chimeric Notchreceptor polypeptide of the present disclosure, when released uponbinding of the first member of the specific binding pair to a secondmember of the specific binding pair, induces production of a geneproduct (a polypeptide; a nucleic acid) in a cell that expresses thechimeric Notch polypeptide. In some cases, the gene product is a nucleicacid. In some cases, the gene product is a polypeptide. Polypeptide geneproducts induced by the released intracellular domain include endogenouspolypeptides (e.g., polypeptides naturally encoded by the cell) andheterologous polypeptides (e.g., polypeptides not naturally encoded bythe cell; polypeptides encoded by a heterologous nucleic acid used togenetically modify the cell). Polypeptide gene products induced by thereleased intracellular domain include secreted polypeptides. Polypeptidegene products induced by the released intracellular domain include cellsurface polypeptides. Polypeptide gene products induced by the releasedintracellular domain include intracellular polypeptides (polypeptidesthat normally are present intracellularly, such as transcriptionfactors). Polypeptide gene products induced by the releasedintracellular domain include receptors, cytokines, hormones, growthfactors, chemokines, cell surface polypeptides, transcription factors(e.g., transcription activators; transcription repressors), apoptosisinducers, apoptosis inhibitors, dominant-negative variants, etc.Polypeptide gene products whose production can be induced by thereleased intracellular domain include transcriptional activators,transcriptional repressors, a chimeric antigen receptor, a T-cellreceptor (TCR), a second chimeric Notch polypeptide, a CAR, atranslation regulator, an immune inhibitory receptor, an immuneinhibitory protein, an immune activating protein, a cytokine receptor, achemokine receptor, a DNA-binding protein, an epigenetic regulator, anRNA-guided endonuclease (e.g., a Cas9 polypeptide), an enzymaticallyinactive Cas9 polypeptide, a site-specific nuclease, a recombinase, atranscription factor that induces differentiation, a transcriptionfactor that induces dedifferentiation, and the like.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of an endogenous gene productin a cell that expresses the chimeric Notch polypeptide. Endogenous geneproducts include, e.g., a chemokine, a chemokine receptor, a cytokine, acytokine receptor, a differentiation factor, a growth factor, a growthfactor receptor, a hormone, a metabolic enzyme, a proliferation inducer,a receptor, a small molecule second messenger synthesis enzyme, a T cellreceptor, a transcription activator, a transcription repressor, atranscriptional activator, a transcriptional repressor, a translationregulator, a translational activator, a translational repressor, anactivating immunoreceptor, an apoptosis in inhibitor, an apoptosisinducer, an immunoactivator, an immunoinhibitor, and an inhibitingimmunoreceptor.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a heterologous gene productin a cell that expresses the chimeric Notch polypeptide. Heterologousgene products include gene products not normally produced by the cell.For example, the cell can be genetically modified with a nucleic acidcomprising a nucleotide sequence encoding a heterologous gene product.Heterologous gene products include, e.g., a chemokine, a chemokinereceptor, a chimeric antigen receptor, a cytokine, a cytokine receptor,a differentiation factor, a growth factor, a growth factor receptor, ahormone, a metabolic enzyme, a pathogen derived protein, a proliferationinducer, a receptor, a RNA guided nuclease, a site-specific nuclease, asmall molecule second messenger synthesis enzyme, a T cell receptor, atoxin derived protein, a transcription activator, a transcriptionrepressor, a transcriptional activator, a transcriptional repressor, atranslation regulator, a translational activator, a translationalrepressor, an activating immunoreceptor, an antibody, an apoptosis ininhibitor, an apoptosis inducer, an engineered T cell receptor, animmunoactivator, an immunoinhibitor, an inhibiting immunoreceptor, anRNA guided DNA binding protein, a T-cell receptor (TCR), a MESApolypeptide, a TANGO polypeptide, and a second synNotch polypeptide(where the second synNotch polypeptide is different from the synNotchpolypeptide whose intracellular domain induced production of the secondsynNotch polypeptide).

Polypeptide gene products that can be induced by the releasedintracellular domain include secreted polypeptides. Non-limitingexamples of secreted polypeptides include, e.g., IL-2, IL-7, TNFalpha,IL-12, GMCSF, EGF, TGFbeta, IL-10, IL-17, IL-4, IL-5, IL-13, IFNalpha,IFNgamma, HMG-B1, secreted PTEN, Wnt, and single chain antibodies.Polypeptide gene products that can be induced by the releasedintracellular domain include dominant negative polypeptides. Examples ofdominant negative polypeptides include, e.g., a dominant negative TGF-βreceptor; a dominant negative variant of STAT3 comprising one or moremutations affecting the DNA binding domain of STAT3 that functions as adominant negative variant; and the like.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a hormone in a cell thatexpresses the chimeric Notch polypeptide. Examples of such hormonesinclude, e.g., erythropoietin (EPO), insulin, secretins, glucagon-likepolypeptide 1 (GLP-1), and the like. Further examples of such hormonesinclude, but are not limited to, activin, inhibin, adiponectin,adipose-derived hormones, adrenocorticotropic hormone, afamelanotide,agouti signaling peptide, allatostatin, amylin, angiotensin, atrialnatriuretic peptide, gastrin, somatotropin, bradykinin, brain-derivedneurotrophic factor, calcitonin, cholecystokinin, ciliary neurotrophicfactor, corticotropin-releasing hormone, cosyntropin, endothelian,enteroglucagon, fibroblast growth factor 15 (FGF15), GFG15/19,follicle-stimulating hormone, gastrin, gastroinhibitory peptide,ghrelin, glucagon, glucagon-like peptide-1, gonadotropin,gonadotropin-releasing hormone, granulocyte-colony-stimulating factor,growth hormone, growth-hormone-releasing hormone, hepcidin, humanchorionic gonadotropin, human placental lactogen, incretin, insulin,insulin analog, insulin aspart, insulin degludec, insulin glargine,insulin lispro, insulin-like growth factor, insulin-like growthfactor-1, insulin-like growth factor-2, leptin, liraglutide, luteinizinghormone, melanocortin, melanocyte-stimulating hormone,alpha-melanocyte-stimulating hormone, melanotin II, minigastrin,N-terminal prohormone of brain natriuretic peptide, nerve growth factor,neurotrophin-3, neurotrophin-4, NPH insulin, obestatin, orexin,osteocalcin, pancreatic hormone, parathyroid hormone, peptide hormone,peptide YY, plasma renin activity, pramlintide, preprohormone,prolactin, relaxin, relaxin family peptide hormone, renin, salcatonin,secretin, secretin family peptide hormone, sincalide, teleost leptins,temporin, tesamorelin, thyroid-stimulating hormone,thyrotropin-releasing hormone, urocortin, urocortin II, urocortin III,vasoactive intestinal peptide, and vitellogenin.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a growth factor in a cellthat expresses the chimeric Notch polypeptide. Examples of such growthfactors include, but are not limited to, hepatocyte stimulating factor,plasmacytoma growth factor, brain derived neurotrophic factor (BDNF),glial derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3),fibroblast growth factor (FGF), transforming growth factor (TGF),platelet transforming growth factor, milk growth factor, endothelialgrowth factors (EGF), endothelial cell-derived growth factors (ECDGF),alpha-endothelial growth factor, beta-endothelial growth factor,neurotrophic growth factor, nerve growth factor (NGF), vascularendothelial growth factor (VEGF), 4-1 BB receptor (4-1BBR), TRAIL(TNF-related apoptosis inducing ligand), artemin (GFRalpha3-RET ligand),BCA-1 (B cell-attracting chemokinel), B lymphocyte chemoattractant(BLC), B cell maturation protein (BCMA), brain-derived neurotrophicfactor (BDNF), bone growth factor such as osteoprotegerin (OPG),bone-derived growth factor, megakaryocyte derived growth factor (MGDF),keratinocyte growth factor (KGF), thrombopoietin, platelet-derivedgrowth factor (PGDF), megakaryocyte derived growth factor (MGDF),keratinocyte growth factor (KGF), platelet-derived growth factor (PGDF),neurotrophin-2 (NT-2), neurotrophin-3 (NT-3), neurotrophin-4 (NT4),neurotrophin-5 (NT-5), glial cell line-derived neurotrophic factor(GDNF), ciliary neurotrophic factor (CNTF), bone Morphogenetic protein 2(BMP2), granulocyte macrophage colony stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), macrophage colonystimulating factor (M-CSF), colony stimulating factor (CSF), and thelike.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a cytokine in a cell thatexpresses the chimeric Notch polypeptide. Examples of such cytokinesinclude, e.g., interferons (e.g., an alpha-interferon, abeta-interferon, a gamma-interferon); interleukins (e.g., IL-1, IL-1α,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 IL-11, IL-12;IL-13, IL-14, IL-15, IL-16, IL-17, IL-17A, IL-18, IL-19, IL-20, IL-24);tumor necrosis factors (e.g., TNF-α); transforming growth factor-beta;TRAIL; and the like. Examples of such cytokines also include flexi-12(Anderson et al. (1997) Hum. Gene Ther. 8:1125), a single chainpolypeptide that combines the two polypeptide chains of an IL-12heterodimer); IL-12 superkine H9 (Levin et al. (2012) Nature 484:529);and the like.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a chemokine in a cell thatexpresses the chimeric Notch polypeptide. Examples of such chemokinesinclude, e.g., MIP-1, MIP-10, MCP-1, RANTES, IP10, and the like.Additional examples of suitable chemokines include, but are not limitedto, chemokine (C-C motif) ligand-2 (CCL2; also referred to as monocytechemotactic protein-1 or MCP1); chemokine (C-C motif) ligand-3 (CCL3;also known as macrophage inflammatory protein-lA or MIP1A); chemokine(C-C motif) ligand-5 (CCL5; also known as RANTES); chemokine (C-C motif)ligand-17 (CCL17; also known as thymus and activation regulatedchemokine or TARC); chemokine (C-C motif) ligand-19 (CCL19; also knownas EBIl ligand chemokine or ELC); chemokine (C-C motif) ligand-21(CCL21; also known as 6Ckine); C-C chemokine receptor type 7 (CCR7);chemokine (C-X-C motif) ligand 9 (CXCL9; also known as monokine inducedby gamma interferon or MIG); chemokine (C-X-C motif) ligand 10 (CXCL10;also known as interferon gamma-induced protein 10 or IP-10); chemokine(C-X-C motif) ligand 11 (CXCL11; also called interferon-inducible T-cellalpha chemoattractant or I-TAC); chemokine (C-X-C motif) ligand 16(CXCL16; chemokine (C motif) ligand (XCL1; also known as lymphotactin);and macrophage colony-stimulating factor (MCSF).

In some cases, the intracellular domain of a binding triggeredtranscriptional switch, e.g., a chimeric Notch receptor polypeptide, ofthe present disclosure, when released upon binding of the first memberof the specific binding pair to a second member of the specific bindingpair, induces production of an antibody in a cell that expresses thechimeric Notch polypeptide. Such antibodies include, e.g., Natalizumab(Tysabri; Biogen Idec/Elan) targeting a4 subunit of a401 anda407integrins (as used in the treatment of MS and Crohn's disease);Vedolizumab (MLN2; Millennium Pharmaceuticals/Takeda) targeting a407integrin (as used in the treatment of UC and Crohn's disease); Belimumab(Benlysta; Human Genome Sciences/GlaxoSmithKline) targeting BAFF (asused in the treatment of SLE); Atacicept (TACI-Ig; Merck/Serono)targeting BAFF and APRIL (as used in the treatment of SLE); Alefacept(Amevive; Astellas) targeting CD2 (as used in the treatment of Plaquepsoriasis, GVHD); Otelixizumab (TRX4; Tolerx/GlaxoSmithKline) targetingCD3 (as used in the treatment of T1D); Teplizumab (MGA031;MacroGenics/Eli Lilly) targeting CD3 (as used in the treatment of TID);Rituximab (Rituxan/Mabthera; Genentech/Roche/Biogen Idec) targeting CD20(as used in the treatment of Non-Hodgkin's lymphoma, RA (in patientswith inadequate responses to TNF blockade) and CLL); Ofatumumab(Arzerra; Genmab/GlaxoSmithKline) targeting CD20 (as used in thetreatment of CLL, RA); Ocrelizumab (2H7; Genentech/Roche/Biogen Idec)targeting CD20 (as used in the treatment of RA and SLE); Epratuzumab(hLL2; Immunomedics/UCB) targeting CD22 (as used in the treatment of SLEand non-Hodgkin's lymphoma); Alemtuzumab (Campath/MabCampath;Genzyme/Bayer) targeting CD52 (as used in the treatment of CLL, MS);Abatacept (Orencia; Bristol-Myers Squibb) targeting CD80 and CD86 (asused in the treatment of RA and JIA, UC and Crohn's disease, SLE);Eculizumab (Soliris; Alexion pharmaceuticals) targeting C5 complementprotein (as used in the treatment of Paroxysmal nocturnalhaemoglobinuria); Omalizumab (Xolair; Genentech/Roche/Novartis)targeting IgE (as used in the treatment of Moderate to severe persistentallergic asthma); Canakinumab (Ilaris; Novartis) targeting IL-1 (as usedin the treatment of Cryopyrin-associated periodic syndromes, SystemicJIA, neonatal-onset multisystem inflammatory disease and acute gout);Mepolizumab (Bosatria; GlaxoSmithKline) targeting IL-5 (as used in thetreatment of Hyper-eosinophilic syndrome); Reslizumab (SCH55700; CeptionTherapeutics) targeting IL-5 (as used in the treatment of Eosinophilicoesophagitis); Tocilizumab (Actemra/RoActemra; Chugai/Roche) targetingIL-6R (as used in the treatment of RA, JIA); Ustekinumab (Stelara;Centocor) targeting IL-12 and IL-23 (as used in the treatment of Plaquepsoriasis, Psoriatic arthritis, Crohn's disease); Briakinumab (ABT-874;Abbott) targeting IL-12 and IL-23 (as used in the treatment of Psoriasisand plaque psoriasis); Etanercept (Enbrel; Amgen/Pfizer) targeting TNF(as used in the treatment of RA, JIA, psoriatic arthritis, AS and plaquepsoriasis); Infliximab (Remicade; Centocor/Merck) targeting TNF (as usedin the treatment of Crohn's disease, RA, psoriatic arthritis, UC, AS andplaque psoriasis); Adalimumab (Humira/Trudexa; Abbott) targeting TNF (asused in the treatment of RA, JIA, psoriatic arthritis, Crohn's disease,AS and plaque psoriasis); Certolizumab pegol (Cimzia; UCB) targeting TNF(as used in the treatment of Crohn's disease and RA); Golimumab(Simponi; Centocor) targeting TNF (as used in the treatment of RA,psoriatic arthritis and AS); and the like. In some cases, the antibodywhose production is induced by the intracellular domain of a synNotchpolypeptide of the present disclosure is a therapeutic antibody for thetreatment of cancer. Such antibodies include, e.g., Ipilimumab targetingCTLA-4 (as used in the treatment of Melanoma, Prostate Cancer, RCC);Tremelimumab targeting CTLA-4 (as used in the treatment of CRC, Gastric,Melanoma, NSCLC); Nivolumab targeting PD-1 (as used in the treatment ofMelanoma, NSCLC, RCC); MK-3475 targeting PD-1 (as used in the treatmentof Melanoma); Pidilizumab targeting PD-1 (as used in the treatment ofHematologic Malignancies); BMS-936559 targeting PD-Li (as used in thetreatment of Melanoma, NSCLC, Ovarian, RCC); MEDI4736 targeting PD-L1;MPDL33280A targeting PD-L1 (as used in the treatment of Melanoma);Rituximab targeting CD20 (as used in the treatment of Non-Hodgkin'slymphoma); Ibritumomab tiuxetan and tositumomab (as used in thetreatment of Lymphoma); Brentuximab vedotin targeting CD30 (as used inthe treatment of Hodgkin's lymphoma); Gemtuzumab ozogamicin targetingCD33 (as used in the treatment of Acute myelogenous leukaemia);Alemtuzumab targeting CD52 (as used in the treatment of Chroniclymphocytic leukaemia); IGN101 and adecatumumab targeting EpCAM (as usedin the treatment of Epithelial tumors (breast, colon and lung));Labetuzumab targeting CEA (as used in the treatment of Breast, colon andlung tumors); huA33 targeting gpA33 (as used in the treatment ofColorectal carcinoma); Pemtumomab and oregovomab targeting Mucins (asused in the treatment of Breast, colon, lung and ovarian tumors); CC49(minretumomab) targeting TAG-72 (as used in the treatment of Breast,colon and lung tumors); cG250 targeting CAIX (as used in the treatmentof Renal cell carcinoma); J591 targeting PSMA (as used in the treatmentof Prostate carcinoma); MOv18 and MORAb-003 (farletuzumab) targetingFolate-binding protein (as used in the treatment of Ovarian tumors);3F8, chl4.18 and KW-2871 targeting Gangliosides (such as GD2, GD3 andGM2) (as used in the treatment of Neuroectodermal tumors and someepithelial tumors); hu3S193 and IgN311 targeting Le y (as used in thetreatment of Breast, colon, lung and prostate tumors); Bevacizumabtargeting VEGF (as used in the treatment of Tumor vasculature); IM-2C6and CDP791 targeting VEGFR (as used in the treatment ofEpithelium-derived solid tumors); Etaracizumab targeting Integrin_V_3(as used in the treatment of Tumor vasculature); Volociximab targetingIntegrin_5_1 (as used in the treatment of Tumor vasculature); Cetuximab,panitumumab, nimotuzumab and 806 targeting EGFR (as used in thetreatment of Glioma, lung, breast, colon, and head and neck tumors);Trastuzumab and pertuzumab targeting ERBB2 (as used in the treatment ofBreast, colon, lung, ovarian and prostate tumors); MM-121 targetingERBB3 (as used in the treatment of Breast, colon, lung, ovarian andprostate, tumors); AMG 102, METMAB and SCH 900105 targeting MET (as usedin the treatment of Breast, ovary and lung tumors); AVE1642, IMC-A12,MK-0646, R1507 and CP 751871 targeting IGF1R (as used in the treatmentof Glioma, lung, breast, head and neck, prostate and thyroid cancer);KB004 and IIIA4 targeting EPHA3 (as used in the treatment of Lung,kidney and colon tumors, melanoma, glioma and haematologicalmalignancies); Mapatumumab (HGS-ETR1) targeting TRAILRI (as used in thetreatment of Colon, lung and pancreas tumors and haematologicalmalignancies); HGS-ETR2 and CS-1008 targeting TRAILR2; Denosumabtargeting RANKL (as used in the treatment of Prostate cancer and bonemetastases); Sibrotuzumab and F19 targeting FAP (as used in thetreatment of Colon, breast, lung, pancreas, and head and neck tumors);81C6 targeting Tenascin (as used in the treatment of Glioma, breast andprostate tumors); Blinatumomab (Blincyto; Amgen) targeting CD3 (as usedin the treatment of ALL); pembrolizumab targeting PD-1 as used in cancerimmunotherapy; 9E10 antibody targeting c-Myc; and the like.

Antibodies that may be expressed, in whole or in part, as the result ofactivation of a binding-triggered transcriptional switch, as describedherein, also include but are not limited to 8H9, Abagovomab, Abciximab,Abituzumab, Abrilumab, Actoxumab, Aducanumab, Afelimomab, Afutuzumab,Alacizumab pegol, ALD518, Alirocumab, Altumomab pentetate, Amatuximab,Anatumomab mafenatox, Anetumab ravtansine, Anifrolumab, Anrukinzumab,Apolizumab, Arcitumomab, Ascrinvacumab, Aselizumab, Atezolizumab,Atinumab, Atlizumab/tocilizumab, Atorolimumab, Bapineuzumab,Basiliximab, Bavituximab, Bectumomab, Begelomab, Benralizumab,Bertilimumab, Besilesomab, Bevacizumab/Ranibizumab, Bezlotoxumab,Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab mertansine, Blosozumab,Bococizumab, Brentuximabvedotin, Brodalumab, Brolucizumab,Brontictuzumab, Cantuzumab mertansine, Cantuzumab ravtansine,Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab,cBR96-doxorubicin immunoconjugate, Cedelizumab, Ch.14.18, Citatuzumabbogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumabtetraxetan, Codrituzumab, Coltuximab ravtansine, Conatumumab,Concizumab, CR6261, Crenezumab, Dacetuzumab, Daclizumab, Dalotuzumab,Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumabmafodotin, Derlotuximab biotin, Detumomab, Dinutuximab, Diridavumab,Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Durvalumab,Dusigitumab, Ecromeximab, Edobacomab, Edrecolomab, Efalizumab,Efungumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab,Emibetuzumab, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol,Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomabcituxetan, Erlizumab, Ertumaxomab, Etrolizumab, Evinacumab, Evolocumab,Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05,Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Firivumab,Flanvotumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab,Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab,Gavilimomab, Gevokizumab, Girentuximab, Glembatumumab vedotin,Gomiliximab, Guselkumab, Ibalizumab, Ibalizumab, Icrucumab,Idarucizumab, Igovomab, IMAB362, Imalumab, Imciromab, Imgatuzumab,Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Inolimomab,Inotuzumab ozogamicin, Intetumumab, Iratumumab, Isatuximab, Itolizumab,Ixekizumab, Keliximab, Lambrolizumab, Lampalizumab, Lebrikizumab,Lemalesomab, Lenzilumab, Lerdelimumab, Lexatumumab, Libivirumab,Lifastuzumab vedotin, Ligelizumab, Lilotomab satetraxetan, Lintuzumab,Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine,Lucatumumab, Lulizumab pegol, Lumiliximab, Lumretuzumab, Margetuximab,Maslimomab, Matuzumab, Mavrilimumab, Metelimumab, Milatuzumab,Minretumomab, Mirvetuximab soravtansine, Mitumomab, Mogamulizumab,Morolimumab, Morolimumab immune, Motavizumab, Moxetumomab pasudotox,Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox,Narnatumab, Nebacumab, Necitumumab, Nemolizumab, Nerelimomab,Nesvacumab, Nofetumomab merpentan, Obiltoxaximab, Obinutuzumab,Ocaratuzumab, Odulimomab, Olaratumab, Olokizumab, Onartuzumab,Ontuxizumab, Opicinumab, Oportuzumab monatox, Orticumab, Otlertuzumab,Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Pankomab,Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab,Patritumab, Perakizumab, Pexelizumab, Pinatuzumab vedotin, Pintumomab,Placulumab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab,Pritumumab, PRO 140, Quilizumab, Racotumomab, Radretumab, Rafivirumab,Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab,Regavirumab, Rilotumumab, Rinucumab, Robatumumab, Roledumab,Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Sacituzumabgovitecan, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab,Seribantumab, Setoxaximab, Sevirumab, SGN-CD19A, SGN-CD33A, Sifalimumab,Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Sofituzumab vedotin,Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab,Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab,Tanezumab, Taplitumomab paptox, Tarextumab, Tefibazumab, Telimomabaritox, Tenatumomab, Teneliximab, Teprotumumab, Tesidolumab, Tetulomab,TGN1412, Ticilimumab/tremelimumab, Tigatuzumab, Tildrakizumab, TNX-650,Toralizumab, Tosatoxumab, Tovetumab, Tralokinumab, TRBS07, Tregalizumab,Trevogrumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab,Ulocuplumab, Urelumab, Urtoxazumab, Vandortuzumab vedotin, Vantictumab,Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Veltuzumab,Vepalimomab, Vesencumab, Visilizumab, Vorsetuzumab mafodotin, Votumumab,Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab, Zolimomab aritox, andthe like.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a neuropeptide in a cellthat expresses the chimeric Notch polypeptide. Examples of suchneuropeptides include, but are not limited to, N-Acetylaspartylglutamicacid, agouti-related peptide, alpha-endorphin, big dynorphin, bombesin,bombesin-like peptides, carbetocin, cocaine-and-amphetamine regulatedtranscript (CART), cholecystokinin, corazonin, corticotropin-likeintermediate peptide, cortistatin, demoxytocin, dynorphin A, dynorphinB, eledoisin, enkephalin, galanin, galanin-like peptide, galmic, galnon,gamma-endorphin, ghrelin, hemopressin, kisspeptin, neurokinin B,neuromedin B, neuromedin N, neuromedin S, neuromedin U, neuromedin S,neuromedin Y, neuropeptide Y, neurotensin, nociceptin, opiorphin,orexin, orexin-A, oxytocin, physalaemin, preprotachykinin, proctolin,proenkephalin, poopiomelanocortin, protein episteme, relaxin-3,somatostatin, substance P, TAC1, tachykinin peptides, vasopressin, andvasotocin.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a transcriptional regulator(e.g., a transcription factor; a transcription inducer; a transcriptionrepressor) in a cell that expresses the chimeric Notch polypeptide.Examples of transcriptional regulators include, e.g., ABT1, ACYP2,AEBP1, AEBP2, AES, AFF1, AFF3, AHR, ANKI, ANK2, ANKFY1, ANKIBI, ANKRD1,ANKRD10, ANKRD2, ANKRD32, ANKRD46, ANKRD49, ANKRD56, ANKRD57, ANKS4B,AR, ARHGAP17, ARID1A, ARIDIB, ARID3A, ARID4A, ARID5B, ARNT, ARNT2,ARNTL, ARNTL2, ARX, ASB10, ASB11, ASB12, ASB15, ASB2, ASB5, ASB8, ASB9,ASH1L, ASH2L, ASXL1, ASZ1, ATF1, ATF3, ATF4, ATF4, ATF5, ATF6, ATF7,ATF7IP, ATM, ATOH1, ATXN3, 1300003B13RIK, B3GAT3, B930041F14RIK, BACH1,BACH2, BARX1, BARX2, BATF, BATF2, BATF3, BAZ2A, BBX, BC003267, BCL11A,BCL11B, BCL3, BCL6, BCL6B, BCLAF1, BCOR, BHLHA15, BHLHE40, BHLHE41,BLZF1, BMYC, BNC1, BNC2, BPNT1, BRCA1, BRWD1, BTBD11, BTF3,6030408C04RIK, CAMK4, CARHSP1, CARM1, CBX4, CBX7, CCNC, CCNH, CCNT1,CCNT2, CDCl₅L, CDK2, CDK4, CDK9, CDKN2C, CDX1, CDX1, CDX2, CEBPA, CEBPB,CEBPD, CEBPG, CEBPG, CEBPZ, CHD4, CHD7, CHGB, CIC, CIITA, CITEDI,CITED2, CITED4, CLOCK, CLPB, CML3, CNOT7, COPS2, CREB1, CREB3, CREB3L1,CREB3L1, CREB3L2, CREB3L3, CREB5, CREBBP, CREBL2, CREM, CSDA, CSDA,CSDC2, CSDE1, CTBP2, CTCF, CTCFL, CTNNB1, CTNNBL1, CXXC1, D11BWG0517E,2300002D11RIK, DACH1, DAXX, DBP, DDIT3, DDX20, DDX54, DDX58, DEAF1, DEK,DIDO1, DLX2, DMRT1, DMRT2, DMRTB1, DNMT1, DNMT3A, DR1, DRG1, DUSP26,DYSFIPI, E2F1, E2F2, E2F3, E2F5, E2F6, EBF1, EBF2, EBF3, EBF3, EED,EGR1, EGR2, EGR3, EHF, EHMT2, EID2, ELAVL2, ELFI, ELF1, ELF2, ELF3,ELF4, ELF5, ELK3, ELK4, ELL2, EMX2, EMX2, EN2, ENPP2, EOMES, EP300,EPAS1, ERF, ERG, ESR1, ESRRA, ESRRB, ESRRG, ETS1, ETS2, ETV1, ETV3,ETV4, ETV5, ETV6, EVIl, EWSR1, EZH1, EZH2, FAH, FBXL10, FBXL11, FBXW7,FEM1A, FEM1B, FEM1C, FHL2, FLII, FMNL2, FOS, FOSB, FOSL1, FOSL2, FOXA1,FOXA2, FOXA3, FOXC1, FOXD1, FOXD2, FOXD3, FOXF1, FOXF1A, FOXF2, FOXG1,FOXII, FOXJ2, FOXJ3, FOXK1, FOXK2, FOXL1, FOXL2, FOXM1, FOXN1, FOXN2,FOXN3, FOXO1, FOXO3, FOXPI, FOXP2, FOXP3, FOXP4, FOXQ1, FUS, FUSIPI,2810021G02RIK, GABPA, GABPB1, GARNL1, GAS7, GATA1, GATA2, GATA3, GATA4,GATA5, GATA5, GATA6, GBX2, GCDH, GCM1, GFI1, GFI1B, GLI2, GLI3, GLIS1,GLIS2, GLIS3, GLS2, GMEB1, GMEB2, GRHL1, GRHL2, GRHL3, GRLF1, GTF2A1,GTF2B, GTF2E2, GTF2F1, GTF2F2, GTF2H2, GTF2H4, GTF2I, GTF2IRD1,GTF2IRD1, GZF1, HAND2, HBP1, HCLS1, HDAC10, HDAC11, HDAC2, HDAC5, HDAC9,HELZ, HES1, HES4, HES5, HES6, HEXIMI, HEY2, HEYL, HHEX, HHEX, HIC1,HIC2, HIF1A, HIF1AN, HIPK2, HIVEPI, HIVEP2, HIVEP2, HIVEP3, HLF, HLTF,HLX, HMBOX1, HMG20A, HMGA2, HMGB2, HMGB3, HNF1B, HNF4A, HNF4G, HOMEZ,HOXA10, HOXA11, HOXA13, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9,HOXB1, HOXB2, HOXB3, HOXB4, HOXB6, HOXB7, HOXB8, HOXB9, HOXC10, HOXC10,HOXCl1, HOXC5, HOXC6, HOXC8, HOXC9, HOXD8, HOXD9, HR, HSBP1, HSF2BP,HTATIP2, HTATSF1, HUWE1, 5830417I10RIK, ID1, ID2, ID3, ID3, IFNAR2,IKBKB, IKBKG, IKZF1, IKZF2, IKZF3, IKZF4, IL31RA, ILF3, INGI, ING2,ING3, ING4, INSM1, INTS12, IQWD1, IRF1, IRF1, IRF2, IRF3, IRF4, IRF5,IRF6, IRF7, IRF8, IRF8, IRX1, IRX2, IRX3, IRX4, IRX5, ISL1, ISL2, ISX,ISX, IVNS1ABP, 2810021J22RIK, JARID1A, JARIDIB, JARIDIC, JARIDID, JDP2,JUN, JUNB, JUND, KLF1, KLF10, KLF11, KLF12, KLF13, KLF15, KLF16, KLF2,KLF3, KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF9, KRR1, 6330416L07RIK,L3MBTL2, LASS2, LASS4, LASS6, LBA1, LBH, LBX1, LCOR, LDB1, LDB2, LEF1,LHX1, LHX2, LHX5, LIMD1, LIN28, LMO1, LMO4, LMX1A, LSM11, LSM4, LYL1,9030612M13RIK, 1810007M14RIK, 3632451006RIK, MAF, MAFA, MAFB, MAFF,MAFG, MAFK, MAGED, MAP3K12, MAPK1, MAPK3, MAPK8, MAPK8IP1, MAX, MAZ,MBD2, MCM2, MCM4, MCM5, MCM6, MCM7, MECOM, MECP2, MED12, MED8, MEF2A,MEF2B, MEF2C, MEF2D, MEIS1, MEIS1, MEIS2, MEOX2, MESP2, MID1, MITF,MKI67IP, MKL1, MLL1, MLL3, MLLT10, MLLT3, MLX, MLXIP, MLXIPL, MNT, MNX1,MPL, MSC, MSRB2, MSX2, MTA3, MTF1, MTF2, MTPN, MXD1, MXD4, MXIl, MYB,MYBBPlA, MYBL2, MYC, MYCBP, MYCL1, MYCN, MYEF2, MYF6, MYNN, MYOCD,MYOD1, MYOG, MYST3, MYST4, MYT1L, MZF1, NAB1, NAB2, NANOG, NARGI, NCOA1,NCOA2, NCOA3, NCOR1, NCOR2, NDN, NEURODI, NEUROD4, NEUROD6, NEUROGI,NEUROG2, NFAT5, NFATC1, NFATC2, NFATC2IP, NFATC3, NFATC3, NFATC4, NFE2,NFE2L1, NFE2L2, NFIA, NFIA, NFIB, NFIC, NFIL3, NFIX, NFKB1, NFKB2,NFKBIB, NFKBIE, NFKBIZ, NFX1, NFXL1, NFYA, NFYB, NHLH1, NKX2-2, NKX2-3,NKX2-5, NKX2-6, NKX6-2, NMI, NOTCHI, NOTCH2, NOTCH3, NOTCH4, NPAS1,NPAS2, NPAS3, NROB1, NROB2, NR1D1, NR1D2, NR1H3, NR1H4, NR1I2, NR1I3,NR2C1, NR2C2, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2,NR4A2, NR4A3, NR5A1, NR5A2, NRARP, NRIP1, NRIP2, NSBP1, NSD1, NUDT12,NULL, NUPRI, 1700065013RIK, OLIGI, OLIG2, OLIG2, ONECUTI, ONECUT2,ONECUT3, ORC2L, OSGINI, OSRI, OSR2, OSTF1, OVOL1, OVOL2, PAPOLA, PAPOLG,PAPPA2, PATZ1, PAWR, PAX2, PAX5, PAX6, PAX7, PAX8, PAX9, PBX1, PBX2,PBX3, PBX4, PCBD1, PCGF6, PDCD11, PDLIM4, PDX1, PEG3, PERI, PFDN1, PGR,PHF1, PHF10, PHF12, PHF13, PHF14, PHF20, PHF21A, PHF5A, PHF7, PHOX2A,PHOX2B, PIAS2, PIR, PITX1, PITX2, PKNOX1, PKNOX2, PLA2G6, PLAGLI,PLAGL2, PLRG1, PML, POGK, POLR2B, POLR2E, POLR2H, POLR3E, POLR3H,POLRMT, POU1F1, POU2AF1, POU2F1, POU2F2, POU3F2, POU3F3, POU3F3, POU5F1,POU6F1, PPARA, PPARD, PPARG, PPARGC1A, PPARGClB, PPP1R12C, PPP1R13B,PPP1R16B, PPP1R1B, PPP2R1A, PPP3CB, PQBP1, PRDM1, PRDM14, PRDM15,PRDM16, PRDM2, PRDM4, PRDM5, PRDM6, PRDM8, PREB, PRKAR1A, PRKCBP1,PROX1, PRRX1, PRRX2, PSMC5, PSMD10, PSMD9, PTF1A, PTGES2, PURB, PWP1,RAB11A, RAB11B, RAB15, RAB18, RAB1B, RAB25, RAB8A, RAB8B, RAI14, RARA,RARB, RARG, RASSF7, RB1, RBBP7, RBL1, RBM14, RBM39, RBM9, RBPJ, RBPJL,RCOR2, REL, RELA, RELB, RERE, REST, REXO4, RFC1, RFX1, RFX2, RFX3, RFX5,RFX7, RFX8, RHOX5, RHOX6, RHOX9, RIPK4, RNF12, RNF14, RNF141, RNF38,RNF4, RORA, RORA, RORB, RORC, RPS6KA4, RREB1, RSRC1, RUNX1, RUNX1T1,RUNX2, RUNX2, RUNX3, RUVBL1, RUVBL2, RXRA, RXRG, RYBP, SAFB2, SALL1,SALL1, SALL2, SALL4, SAP30, SAP30BP, SATB1, SATB2, SATB2, SCANDI, SCAP,SCRT2, SEC14L2, SERTADI, SF1, SFPIl, SFRS5, SH3D19, SH3PXD2B, SHANK3,SHOX2, SHPRH, SIN3A, SIN3B, SIRT2, SIRT3, SIRT5, SIX1, SIX1, SIX2, SIX3,SIX4, SIX5, SKI, SMAD1, SMAD2, SMAD3, SMAD7, SMARCA1, SMARCA2, SMARCA5,SMARCB1, SMYD1, SNAIl, SNAI2, SNAPC2, SNAPC4, SNIPI, SOLH, SOX1, SOX10,SOX11, SOX12, SOX13, SOX15, SOX17, SOX18, SOX2, SOX21, SOX4, SOX5, SOX6,SOX7, SOX8, SOX9, SP1, SP110, SP140L, SP2, SP3, SP4, SP6, SP8, SPDEF,SPEN, SPIl, SPIB, SQSTM1, SREBF1, SREBF2, SREBF2, SRF, SSBP2, SSBP3,SSBP4, SSRP1, ST18, STAG1, STAT1, STAT1, STAT2, STAT3, STAT4, STAT5A,STAT5B, STAT5B, STAT6, SUB1, SUZ12, TADA2L, TAF13, TAF5, TAF5L, TAF7,TAF9, TAL, TALl, TARDBP, TBPL1, TBR1, TBX1, TBX10, TBX15, TBX18, TBX2,TBX2, TBX20, TBX21, TBX3, TBX4, TBX5, TBX6, TCEA1, TCEA3, TCEAL1, TCEB3,TCERG1, TCF12, TCF15, TCF19, TCF20, TCF21, TCF21, TCF3, TCF4, TCF7,TCF7L2, TCFAP2A, TCFAP2B, TCFAP2C, TCFCP2L1, TCFE2A, TCFE3, TCFEB,TCFEC, TCFL5, TEAD1, TEAD2, TEAD3, TEAD4, TEF, TFAP2A, TFAP2C, TFCP2L1,TFDP2, TFEB, TFEC, TGFB1I1, TGIF1, TGIF2, TGIF2LX, THRA, THRAP3, THRB,THRSP, TIAL1, TLE1, TLE6, TMEM131, TMPO, TNFAIP3, TOB1, TOX4, TP63,TRERFI, TRIB3, TRIM24, TRIM28, TRIM30, TRIP13, TRIP4, TRIP6, TRP53,TRP53BP1, TRP63, TRPS1, TRPS1, TSC22D1, TSC22D2, TSC22D3, TSC22D4,TSHZ1, TSHZ1, TSHZ3, TTRAP, TUB, TULP4, TWISTI, TWIST2, TYSND1, UBE2W,UBN1, UBP1, UBTF, UGP2, UHRF1, UHRF2, UNCX, USF1, USF2, UTF1, VDR,VEZF1, VGLL2, VSX1, WASL, WHSC1, WHSC2, WT1, WWP1, WWTR1, XBP1, YAF2,YY1, ZBED1, ZBED4, ZBTB1, ZBTB10, ZBTB16, ZBTB16, ZBTB17, ZBTB2, ZBTB20,ZBTB22, ZBTB25, ZBTB32, ZBTB38, ZBTB4, ZBTB43, ZBTB45, ZBTB47, ZBTB7A,ZBTB7B, ZBTB7C, ZCCHC8, ZDHHC13, ZDHHC16, ZDHHC21, ZDHHC5, ZDHHC6, ZEB2,ANK2ZEB2, ZFHX2, ZFHX3, ZFHX4, ZFP105, ZFP110, ZFP143, ZFP148, ZFP161,ZFP192, ZFP207, ZFP219, ZFP238, ZFP263, ZFP275, ZFP277, ZFP281, ZFP287,ZFP292, ZFP35, ZFP354C, ZFP36, ZFP36L1, ZFP386, ZFP407, ZFP42, ZFP423,ZFP426, ZFP445, ZFP451, ATF5ZFP451, ZFP467, ZFP52, ZFP57, ZFP592,ZFP593, ZFP597, ZFP612, ZFP637, ZFP64, ZFP647, ZFP748, ZFP810, ZFP9,ZFP91, ZFPM1, ZFPM2, ZFX, ZHX2, ZHX3, ZIC1, ZIC2, ZIC3, ZIC4, ZIC5,ZKSCAN1, ZKSCAN3, ZMYND11, ZNF143, ZNF160, ZNF175, ZNF184, ZNF192,ZNF213, ZNF217, ZNF219, ZNF22, ZNF238, ZNF24, ZNF267, ZNF273, ZNF276,ZNF280D, ZNF281, ZNF292, ZNF311, ZNF331, ZNF335, ZNF337, ZNF33B, ZNF366,ZNF394, ZNF398, ZNF41, ZNF410, ZNF415, ZNF423, ZNF436, ZNF444, ZNF445,ZNF451, ZNF460, ZNF496, ZNF498, ZNF516, ZNF521, ZNF532, ZNF536, ZNF546,ZNF552, ZNF563, ZNF576, ZNF580, ZNF596, ZNF621, ZNF628, ZNF648, ZNF649,ZNF652, ZNF655, ZNF664, ZNF668, ZNF687, ZNF692, ZNF696, ZNF697, ZNF710,ZNF80, ZNF91, ZNF92, ZNRD1, ZSCAN10, ZSCAN16, ZSCAN20, ZSCAN21, ZXDC,and ZZZ3. Additional examples of transcriptional regulators are asdescribed above. Non-limiting examples of transcription factors(transcriptional activators; transcriptional repressors) are depicted inFIGS. 37-83. For example, a transcription factor can comprise an aminoacid sequence having at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence depicted in any one ofFIGS. 37-83.

Additional examples of transcriptional regulators as described aboveinclude but are not limited to transcription factors having a regulatoryrole in one or more immune cells (i.e., immune cell regulatorytranscription factors). Suitable immune cell regulatory transcriptionfactors include, e.g., 2210012G02Rik, Akap81, Appl2, Arid4b, Arid5b,Ashl1, Atf7, Atm, C430014K11Rik, Chd9, Dmtfl, Fos, Foxol, Foxp1, Hmboxl,Kdm5b, Klf2, Mga, M1, Mll3, Myst4, Pcgf6, Rev31, Scml4, Scp2, Smarca2,Ssbp2, Suhw4, Tcf7, Tfdp2, Tox, Zbtb20, Zbtb44, Zeb1, Zfm1, Zfp1,Zfp319, Zfp329, Zfp35, Zfp386, Zfp445, Zfp518, Zfp652, Zfp827, Zhx2,Eomes, Arnt1, Bbx, Hbpl, Jun, Mef2d, Mterfd1, Nfat5, Nfe212, Nrld2,Phf21a, Taf4b, Trf, Zbtb25, Zfp326, Zfp451, Zfp58, Zfp672, Egr2, Ikzf2,Tafld, Chrac, Dnajb6, Aplp2, Batf, Bhlhe40, Fosb, Hist1h1c, Hopx, Ifih1,Ikzf3, Lass4, Lin54, Mxd1, Mxi1, Prdml, Prfl, Rora, Rpa2, Sap30, Stat2,Stat3, Taf9b, Tbx21, Trpsl, Xbpl, Zeb2, Atf3, Cenpc1, Lass6, Rbl,Zbtb41, Crem, Fos12, Gtf2b, Irf7, Maff, Nr4a1, Nr4a2, Nr4a3, Obfc2a,Rbl2, Rel, Rybp, Sral, Tgif1, Tnfaip3, Uhrf2, Zbtbl, Ccdc124, Csda,E2f3, Epas1, HIf, H2afz, Hif1a, Ikzf5, Irf4, Nsbp1, Pim1, Rfc2, Swap70,Tfblm, 2610036L11Rik, 5133400G04Rik, Apitdl, Blm, Brca1, Brip1, Cld,C79407, Cenpa, Cfl1, Clspn, Ddx1, Dscc1, E2, E2f8, Ercc61, Ezh2, Fen1,Foxm1, Gen1, Gsg2, H2afx, Hdac1, Hdgf, Hells, Hist1h1e, Hist3h2a, Hjurp,Hmgb2, Hmgb3, Irf1, Irf8, Kif22, Kif4, Lig1, Lmo2, Lnp, Mbd4, Mcm2,Mcm3, Mcm4, Mcm5, Mcm6, Mcm7, Mybl2, Neil3, Nusap1, Orc61, Pola1, Pola2,Pole, Pole2, Polh, Polr2f, Polr2j, Ppplr8, Prim2, Psmc3ip, Rad51,Rad51c, Rad541, Rfc3, Rfc4, Rnps1, Rpa1, Smarcc1, Spic, Ssrp1, Taf9,Tfdp1, Tmpo, Topbp1, Trdmt1, Uhrf1, Wdhd1, Whsc1, Zbp1, Zbtb32, Zfp367,Carl, Polg2, Atr, LefI, Myc, Nucb2, Satb1, Taf1a, Ift57, Apex1, Chd7,Chtf8, Ctnnb1, Etv3, Irf9, Myb, Mybbp1a, Pms2, Preb, Sp110, Stat1,Trp53, Zfp414, App, Cdk9, Ddb1, Hsf2, Lbr, Pa2g4, Rbms1, Rfc1, Rfc5,Tada21, Tex261, Xrcc6, and the like.

In some cases, a transcription factor may be an artificial transcriptionfactor (ATF) including but not limited to e.g., Zinc-finger-basedartificial transcription factors (including e.g., those described inSera T. Adv Drug Deliv Rev. 2009 61(7-8):513-26; Collins et al. CurrOpin Biotechnol. 2003 14(4):371-8; Onori et al. BMC Mol Biol. 2013 14:3the disclosures of which are incorporated herein by reference in theirentirety).

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of an immunoreceptor (e.g., anactivating immunoreceptor or an inhibitory immunoreceptor) in a cellthat expresses the chimeric Notch polypeptide. Examples of suchimmunoreceptors include activating immunoreceptors. A suitableactivating immunoreceptor can comprise an immunoreceptor tyrosine-basedactivation motif (ITAM). An ITAM motif is YX₁X₂L/I, where X₁ and X₂ areindependently any amino acid. A suitable immunoreceptor can comprise anITAM motif-containing portion that is derived from a polypeptide thatcontains an ITAM motif. For example, a suitable immunoreceptor cancomprise an ITAM motif-containing domain from any ITAM motif-containingprotein. Thus, a suitable immunoreceptor need not contain the entiresequence of the entire protein from which it is derived. Examples ofsuitable ITAM motif-containing polypeptides include, but are not limitedto: DAP12; FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta);CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3Z (CD3 zeta); and CD79A(antigen receptor complex-associated protein alpha chain). Furtherexamples of suitable ITAM motif-containing polypeptides are as describedabove.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a T-cell surfaceglycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3complex); OKT3, delta chain; T-cell receptor T3 delta chain; T-cellsurface glycoprotein CD3 delta chain; etc.) in a cell that expresses thechimeric Notch polypeptide. In some cases, the intracellular domain of achimeric Notch receptor polypeptide of the present disclosure, whenreleased upon binding of the first member of the specific binding pairto a second member of the specific binding pair, induces production of aT-cell surface glycoprotein CD3 epsilon chain (also known as CD3e,T-cell surface antigen T3/Leu-4 epsilon chain, T-cell surfaceglycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.) ina cell that expresses the chimeric Notch polypeptide.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a co-stimulatorypolypeptide in a cell that expresses the chimeric Notch polypeptide.Non-limiting examples of suitable co-stimulatory polypeptides include,but are not limited to, 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27,CD30, GITR, and HVEM. Further examples of suitable co-stimulatorypolypeptides are as described above.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of an inhibitoryimmunoreceptor in a cell that expresses the chimeric Notch polypeptide.An inhibitory immunoreceptor can comprise an immunoreceptortyrosine-based inhibition motif (ITIM), an immunoreceptor tyrosine-basedswitch motif (ITSM), an NpxY motif, or a YXX® motif. Suitable inhibitorimmunoreceptors include PD1; CTLA4; BTLA; CD160; KRLG-1; 2B4; Lag-3; andTim-3. See, e.g., Odorizzi and Wherry (2012) J. Immunol. 188:2957; andBaitsch et al. (2012) PLoS One 7:e30852. Further examples of inhibitoryimmunoreceptors are as described above.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a recombinase in a cellthat expresses the chimeric Notch polypeptide. Non-limiting examples ofrecombinases include a Cre recombinase; a Flp recombinase; a Drerecombinase; and the like. A further example of a recombinase is a FLPerecombinase (see, e.g., Akbudak and Srivastava (2011) Mol. Biotechnol.49:82). A suitable recombinase is a Flpo recombinase. Further examplesof recombinases are as described above.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a site-specific nuclease ina cell that expresses the chimeric Notch polypeptide. Non-limitingexamples of site-specific nucleases include, but are not limited to, anRNA-guided DNA binding protein having nuclease activity, e.g., a Cas9polypeptide; a transcription activator-like effector nuclease (TALEN);Zinc-finger nucleases; and the like. Further examples of site-specificnucleases are as described above.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of an apoptosis inducer in acell that expresses the chimeric Notch polypeptide. Non-limitingexamples of apoptosis inducers are tBID polypeptides. The term “tBID”refers to the C-terminal truncated fragment of the BH3 interacting deathagonist (BID) protein which results from the enzymatic cleavage ofcytosolic BID (e.g., by active caspase). At an early stage of apoptosis,tBID translocates to the mitochondria and mediates the release of Cyt ctherefrom. Non-limiting examples of tBID proteins include human tBID(amino acids 61-195 of the amino acid sequence provided in GenBankAccession No. CAG30275).

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a binding-triggeredtranscriptional switch in a cell that expresses the chimeric Notchpolypeptide.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a TCR in a cell thatexpresses the chimeric Notch polypeptide. The TCR is in some casesspecific for an epitope of an antigen. Examples of such antigensinclude, e.g., tumor antigens; cancer cell-associated antigens;hematological malignancy antigens; solid tumor antigens; cell surfaceantigens (e.g., cell surface antigens targeted by a T cell receptor(TCR); intracellular antigens; and the like. Examples of hematologicalmalignancy antigens include, e.g., CD19 (as expressed in e.g., B-cells),CD20 (as expressed in e.g., B-cells), CD22 (as expressed in e.g.,B-cells), CD30 (as expressed in e.g., B-cells), CD33 (as expressed ine.g., Myeloid cells), CD70 (as expressed in e.g., B-cell/T-cells), CD123(as expressed in e.g., Myeloid cells), Kappa (as expressed in e.g.,B-cells), Lewis Y (as expressed in e.g., Myeloid cells), NKG2D ligands(as expressed in e.g., Myeloid cells), RORI (as expressed in e.g.,B-cells), SLAMF7/CS1 (as expressed in e.g., myeloma cells, naturalkiller cells, T cells, and most B-cell types), CD138 (as expressed ine.g., malignant plasma cells in multiple myelomas), CD56 (as expressedin e.g., myeloma cells, neural cells, natural killer cells, T cells, andtrabecular osteoblasts) CD38 (as expressed in e.g., B-cell/T-cells) andCD160 (as expressed in e.g., NK cells/T-cells), and the like. Examplesof solid tumor antigens include, e.g., B7H3 (as expressed in e.g.,Sarcoma, glioma), CAIX (as expressed in e.g., Kidney), CD44 v6/v7 (asexpressed in e.g., Cervical), CD171 (as expressed in e.g.,Neuroblastoma), CEA (as expressed in e.g., Colon), EGFRvIII (asexpressed in e.g., Glioma), EGP2 (as expressed in e.g., Carcinomas),EGP40 (as expressed in e.g., Colon), EphA2 (as expressed in e.g.,Glioma, lung), ErbB2(HER2) (as expressed in e.g., Breast, lung,prostate, glioma), ErbB receptor family (as expressed in e.g., Breast,lung, prostate, glioma), ErbB3/4 (as expressed in e.g., Breast,ovarian), HLA-A1/MAGE1 (as expressed in e.g., Melanoma), HLA-A2/NY-ESO-1(as expressed in e.g., Sarcoma, melanoma), FR-a (as expressed in e.g.,Ovarian), FAP† (as expressed in e.g., Cancer associated fibroblasts),FAR (as expressed in e.g., Rhabdomyosarcoma), GD2 (as expressed in e.g.,Neuroblastoma, sarcoma, melanoma), GD3 (as expressed in e.g., Melanoma,lung cancer), HMW-MAA (as expressed in e.g., Melanoma), IL11Ra (asexpressed in e.g., Osteosarcoma), IL13Ra2 (as expressed in e.g.,Glioma), Lewis Y (as expressed in e.g., Breast/ovarian/pancreatic),Mesothelin (as expressed in e.g., Mesothelioma, breast, pancreas), Muc1(as expressed in e.g., Ovarian, breast, prostate), NCAM (as expressed ine.g., Neuroblastoma, colorectal), NKG2D ligands (as expressed in e.g.,Ovarian, sacoma), PSCA (as expressed in e.g., Prostate, pancreatic),PSMA (as expressed in e.g., Prostate), TAG72 (as expressed in e.g.,Colon), VEGFR-2 (as expressed in e.g., Tumor vasculature), Axl (asexpressed in e.g., Lung cancer), Met (as expressed in e.g., Lungcancer), α5β3 (as expressed in e.g., Tumor vasculature), α5β1 (asexpressed in e.g., Tumor vasculature), TRAIL-R/TRAIL-R2 (as expressed ine.g., Solid tumors (colon, lung, pancreas) and hematologicalmalignancies), RANKL (as expressed in e.g., Prostate cancer and bonemetastases), Tenacin (as expressed in e.g., Glioma, epithelial tumors(breast, prostate)), EpCAM (as expressed in e.g., Epithelial tumors(breast, colon, lung)), CEA (as expressed in e.g., Epithelial tumors(breast, colon, lung)), gpA33 (as expressed in e.g., Colorectalcarcinoma), Mucins (as expressed in e.g., Epithelial tumors (breast,colon, lung, ovarian)), TAG-72 (as expressed in e.g., Epithelial tumors(breast, colon, lung)), EphA3 (as expressed in e.g., Lung, kidney,melanoma, glioma, hematological malignancies) and IGF1R (as expressed ine.g., Lung, breast, head and neck, prostate, thyroid, glioma). Examplesof surface and intracellular antigens include, e.g., Her2 (gene symbolERBB2), MAGE-A1 (gene symbol MAGEA1), MART-1 (gene symbol MLANA), NY-ESO(gene symbol CTAG1), WT1 (gene symbol WT1), MUC17 and MUC13. Examples ofother antigens include, e.g., BCMA (gene symbol TNFRSF17), B7H6 (genesymbol NCR3LG1), CAIX (gene symbol CA9), CD123 (gene symbol IL3RA),CD138 (gene symbol SDC1), CD171 (gene symbol L1CAM), CD19 (gene symbolCD19), CD20 (gene symbol CD20), CD22 (gene symbol CD22), CD30 (genesymbol TNFRSF8), CD33 (gene symbol CD33), CD38 (gene symbol CD38), CD44,splice variants incl 7 and 8 (denoted vX in literature) (gene symbolCD44), CEA, CS1 (gene symbol SLAMF7), EGFRvIII (gene symbol EGFR, vIIIdeletion variant), EGP2, EGP40 (gene symbol EPCAM), Erb family member(gene symbol ERBB1, ERBB2, ERBB3, ERBB4), FAP (gene symbol FAP), fetalacetylcholine receptor (gene symbol AChR), Folate receptor alpha (genesymbol FOLR1), Folate receptor beta (gene symbol FOLR2), GD2, GD3, GPC3(gene symbol GPC3), Her2/neu (gene symbol ERBB2), IL-13Ra2 (gene symbolIL13RA2), Kappa light chain (gene symbol IGK), Lewis-Y, Mesothelin (genesymbol MSLN), Mucin-1 (gene symbol MUC1), Mucin-16 (gene symbol MUC16),NKG2D ligands, prostate specific membrane antigen (PSMA) (gene symbolFOLH1), prostate stem cell antigen (PSCA) (gene symbol PSCA), receptortyrosine kinase-like orphan receptor 1 (gene symbol ROR1), andAnaplastic Lymphoma Receptor Tyrosine Kinase (gene symbol ALK).

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a MESA polypeptide in acell that expresses the chimeric Notch polypeptide. The MESA polypeptidein some cases comprises a domain that specifically binds an antigen.Examples of such antigens include, e.g., tumor antigens; cancercell-associated antigens; hematological malignancy antigens; solid tumorantigens; cell surface antigens (e.g., cell surface antigens targeted bya T cell receptor (TCR); intracellular antigens; and the like. Examplesof hematological malignancy antigens include, e.g., CD19 (as expressedin e.g., B-cells), CD20 (as expressed in e.g., B-cells), CD22 (asexpressed in e.g., B-cells), CD30 (as expressed in e.g., B-cells), CD33(as expressed in e.g., Myeloid cells), CD70 (as expressed in e.g.,B-cell/T-cells), CD123 (as expressed in e.g., Myeloid cells), Kappa (asexpressed in e.g., B-cells), Lewis Y (as expressed in e.g., Myeloidcells), NKG2D ligands (as expressed in e.g., Myeloid cells), RORI (asexpressed in e.g., B-cells), SLAMF7/CS1 (as expressed in e.g., myelomacells, natural killer cells, T cells, and most B-cell types), CD138 (asexpressed in e.g., malignant plasma cells in multiple myelomas), CD56(as expressed in e.g., myeloma cells, neural cells, natural killercells, T cells, and trabecular osteoblasts) CD38 (as expressed in e.g.,B-cell/T-cells) and CD160 (as expressed in e.g., NK cells/T-cells), andthe like. Examples of solid tumor antigens include, e.g., B7H3 (asexpressed in e.g., Sarcoma, glioma), CAIX (as expressed in e.g.,Kidney), CD44 v6/v7 (as expressed in e.g., Cervical), CD171 (asexpressed in e.g., Neuroblastoma), CEA (as expressed in e.g., Colon),EGFRvIII (as expressed in e.g., Glioma), EGP2 (as expressed in e.g.,Carcinomas), EGP40 (as expressed in e.g., Colon), EphA2 (as expressed ine.g., Glioma, lung), ErbB2(HER2) (as expressed in e.g., Breast, lung,prostate, glioma), ErbB receptor family (as expressed in e.g., Breast,lung, prostate, glioma), ErbB3/4 (as expressed in e.g., Breast,ovarian), HLA-A1/MAGE1 (as expressed in e.g., Melanoma), HLA-A2/NY-ESO-1(as expressed in e.g., Sarcoma, melanoma), FR-a (as expressed in e.g.,Ovarian), FAP† (as expressed in e.g., Cancer associated fibroblasts),FAR (as expressed in e.g., Rhabdomyosarcoma), GD2 (as expressed in e.g.,Neuroblastoma, sarcoma, melanoma), GD3 (as expressed in e.g., Melanoma,lung cancer), HMW-MAA (as expressed in e.g., Melanoma), IL11Ra (asexpressed in e.g., Osteosarcoma), IL13Ra2 (as expressed in e.g.,Glioma), Lewis Y (as expressed in e.g., Breast/ovarian/pancreatic),Mesothelin (as expressed in e.g., Mesothelioma, breast, pancreas), Muc(as expressed in e.g., Ovarian, breast, prostate), NCAM (as expressed ine.g., Neuroblastoma, colorectal), NKG2D ligands (as expressed in e.g.,Ovarian, sacoma), PSCA (as expressed in e.g., Prostate, pancreatic),PSMA (as expressed in e.g., Prostate), TAG72 (as expressed in e.g.,Colon), VEGFR-2 (as expressed in e.g., Tumor vasculature), Axl (asexpressed in e.g., Lung cancer), Met (as expressed in e.g., Lungcancer), α5β3 (as expressed in e.g., Tumor vasculature), α5β1 (asexpressed in e.g., Tumor vasculature), TRAIL-R/TRAIL-R2 (as expressed ine.g., Solid tumors (colon, lung, pancreas) and hematologicalmalignancies), RANKL (as expressed in e.g., Prostate cancer and bonemetastases), Tenacin (as expressed in e.g., Glioma, epithelial tumors(breast, prostate)), EpCAM (as expressed in e.g., Epithelial tumors(breast, colon, lung)), CEA (as expressed in e.g., Epithelial tumors(breast, colon, lung)), gpA33 (as expressed in e.g., Colorectalcarcinoma), Mucins (as expressed in e.g., Epithelial tumors (breast,colon, lung, ovarian)), TAG-72 (as expressed in e.g., Epithelial tumors(breast, colon, lung)), EphA3 (as expressed in e.g., Lung, kidney,melanoma, glioma, hematological malignancies) and IGF1R (as expressed ine.g., Lung, breast, head and neck, prostate, thyroid, glioma). Examplesof surface and intracellular antigens include, e.g., Her2 (gene symbolERBB2), MAGE-A1 (gene symbol MAGEA1), MART-1 (gene symbol MLANA), NY-ESO(gene symbol CTAG1), WT1 (gene symbol WT1), MUC17 and MUC13. Examples ofother antigens include, e.g., BCMA (gene symbol TNFRSF17), B7H6 (genesymbol NCR3LG1), CAIX (gene symbol CA9), CD123 (gene symbol IL3RA),CD138 (gene symbol SDC1), CD171 (gene symbol L1CAM), CD19 (gene symbolCD19), CD20 (gene symbol CD20), CD22 (gene symbol CD22), CD30 (genesymbol TNFRSF8), CD33 (gene symbol CD33), CD38 (gene symbol CD38), CD44,splice variants incl 7 and 8 (denoted vX in literature) (gene symbolCD44), CEA, CS1 (gene symbol SLAMF7), EGFRvIII (gene symbol EGFR, vIIIdeletion variant), EGP2, EGP40 (gene symbol EPCAM), Erb family member(gene symbol ERBB1, ERBB2, ERBB3, ERBB4), FAP (gene symbol FAP), fetalacetylcholine receptor (gene symbol AChR), Folate receptor alpha (genesymbol FOLR1), Folate receptor beta (gene symbol FOLR2), GD2, GD3, GPC3(gene symbol GPC3), Her2/neu (gene symbol ERBB2), IL-13Ra2 (gene symbolIL13RA2), Kappa light chain (gene symbol IGK), Lewis-Y, Mesothelin (genesymbol MSLN), Mucin-1 (gene symbol MUC1), Mucin-16 (gene symbol MUC16),NKG2D ligands, prostate specific membrane antigen (PSMA) (gene symbolFOLH1), prostate stem cell antigen (PSCA) (gene symbol PSCA), receptortyrosine kinase-like orphan receptor 1 (gene symbol RORI), andAnaplastic Lymphoma Receptor Tyrosine Kinase (gene symbol ALK).

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a CAR in a cell thatexpresses the chimeric Notch polypeptide. The CAR in some casescomprises a domain that specifically binds an antigen. Examples of suchantigens include, e.g., tumor antigens; cancer cell-associated antigens;hematological malignancy antigens; solid tumor antigens; cell surfaceantigens (e.g., cell surface antigens targeted by a T cell receptor(TCR); intracellular antigens; and the like. Examples of hematologicalmalignancy antigens include, e.g., CD19 (as expressed in e.g., B-cells),CD20 (as expressed in e.g., B-cells), CD22 (as expressed in e.g.,B-cells), CD30 (as expressed in e.g., B-cells), CD33 (as expressed ine.g., Myeloid cells), CD70 (as expressed in e.g., B-cell/T-cells), CD123(as expressed in e.g., Myeloid cells), Kappa (as expressed in e.g.,B-cells), Lewis Y (as expressed in e.g., Myeloid cells), NKG2D ligands(as expressed in e.g., Myeloid cells), RORI (as expressed in e.g.,B-cells), SLAMF7/CS1 (as expressed in e.g., myeloma cells, naturalkiller cells, T cells, and most B-cell types), CD138 (as expressed ine.g., malignant plasma cells in multiple myelomas), CD56 (as expressedin e.g., myeloma cells, neural cells, natural killer cells, T cells, andtrabecular osteoblasts) CD38 (as expressed in e.g., B-cell/T-cells) andCD160 (as expressed in e.g., NK cells/T-cells), and the like. Examplesof solid tumor antigens include, e.g., B7H3 (as expressed in e.g.,Sarcoma, glioma), CAIX (as expressed in e.g., Kidney), CD44 v6/v7 (asexpressed in e.g., Cervical), CD171 (as expressed in e.g.,Neuroblastoma), CEA (as expressed in e.g., Colon), EGFRvIII (asexpressed in e.g., Glioma), EGP2 (as expressed in e.g., Carcinomas),EGP40 (as expressed in e.g., Colon), EphA2 (as expressed in e.g.,Glioma, lung), ErbB2(HER2) (as expressed in e.g., Breast, lung,prostate, glioma), ErbB receptor family (as expressed in e.g., Breast,lung, prostate, glioma), ErbB3/4 (as expressed in e.g., Breast,ovarian), HLA-A1/MAGE1 (as expressed in e.g., Melanoma), HLA-A2/NY-ESO-1(as expressed in e.g., Sarcoma, melanoma), FR-a (as expressed in e.g.,Ovarian), FAP† (as expressed in e.g., Cancer associated fibroblasts),FAR (as expressed in e.g., Rhabdomyosarcoma), GD2 (as expressed in e.g.,Neuroblastoma, sarcoma, melanoma), GD3 (as expressed in e.g., Melanoma,lung cancer), HMW-MAA (as expressed in e.g., Melanoma), IL11Ra (asexpressed in e.g., Osteosarcoma), IL13Ra2 (as expressed in e.g.,Glioma), Lewis Y (as expressed in e.g., Breast/ovarian/pancreatic),Mesothelin (as expressed in e.g., Mesothelioma, breast, pancreas), Muc(as expressed in e.g., Ovarian, breast, prostate), NCAM (as expressed ine.g., Neuroblastoma, colorectal), NKG2D ligands (as expressed in e.g.,Ovarian, sacoma), PSCA (as expressed in e.g., Prostate, pancreatic),PSMA (as expressed in e.g., Prostate), TAG72 (as expressed in e.g.,Colon), VEGFR-2 (as expressed in e.g., Tumor vasculature), Axl (asexpressed in e.g., Lung cancer), Met (as expressed in e.g., Lungcancer), α5β3 (as expressed in e.g., Tumor vasculature), α5β1 (asexpressed in e.g., Tumor vasculature), TRAIL-R1/TRAIL-R2 (as expressedin e.g., Solid tumors (colon, lung, pancreas) and hematologicalmalignancies), RANKL (as expressed in e.g., Prostate cancer and bonemetastases), Tenacin (as expressed in e.g., Glioma, epithelial tumors(breast, prostate)), EpCAM (as expressed in e.g., Epithelial tumors(breast, colon, lung)), CEA (as expressed in e.g., Epithelial tumors(breast, colon, lung)), gpA33 (as expressed in e.g., Colorectalcarcinoma), Mucins (as expressed in e.g., Epithelial tumors (breast,colon, lung, ovarian)), TAG-72 (as expressed in e.g., Epithelial tumors(breast, colon, lung)), EphA3 (as expressed in e.g., Lung, kidney,melanoma, glioma, hematological malignancies) and IGF1R (as expressed ine.g., Lung, breast, head and neck, prostate, thyroid, glioma). Examplesof surface and intracellular antigens include, e.g., Her2 (gene symbolERBB2), MAGE-A1 (gene symbol MAGEA1), MART-1 (gene symbol MLANA), NY-ESO(gene symbol CTAG1), WT1 (gene symbol WT1), MUC17 and MUC13. Examples ofother antigens include, e.g., BCMA (gene symbol TNFRSF17), B7H6 (genesymbol NCR3LG1), CAIX (gene symbol CA9), CD123 (gene symbol IL3RA),CD138 (gene symbol SDC1), CD171 (gene symbol L1CAM), CD19 (gene symbolCD19), CD20 (gene symbol CD20), CD22 (gene symbol CD22), CD30 (genesymbol TNFRSF8), CD33 (gene symbol CD33), CD38 (gene symbol CD38), CD44,splice variants incl 7 and 8 (denoted vX in literature) (gene symbolCD44), CEA, CS1 (gene symbol SLAMF7), EGFRvIII (gene symbol EGFR, vIIIdeletion variant), EGP2, EGP40 (gene symbol EPCAM), Erb family member(gene symbol ERBB1, ERBB2, ERBB3, ERBB4), FAP (gene symbol FAP), fetalacetylcholine receptor (gene symbol AChR), Folate receptor alpha (genesymbol FOLR1), Folate receptor beta (gene symbol FOLR2), GD2, GD3, GPC3(gene symbol GPC3), Her2/neu (gene symbol ERBB2), IL-13Ra2 (gene symbolIL13RA2), Kappa light chain (gene symbol IGK), Lewis-Y, Mesothelin (genesymbol MSLN), Mucin-1 (gene symbol MUC1), Mucin-16 (gene symbol MUC16),NKG2D ligands, prostate specific membrane antigen (PSMA) (gene symbolFOLH1), prostate stem cell antigen (PSCA) (gene symbol PSCA), receptortyrosine kinase-like orphan receptor 1 (gene symbol RORI), andAnaplastic Lymphoma Receptor Tyrosine Kinase (gene symbol ALK).

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide (e.g., a first synNotch polypeptide) of the presentdisclosure, when released upon binding of the first member of thespecific binding pair to a second member of the specific binding pair,induces production of a second synNotch polypeptide in a cell thatexpresses the chimeric Notch polypeptide. The second synNotchpolypeptide in some cases comprises a domain that specifically binds anantigen. Examples of such antigens include, e.g., tumor antigens; cancercell-associated antigens; hematological malignancy antigens; solid tumorantigens; cell surface antigens (e.g., cell surface antigens targeted bya T cell receptor (TCR); intracellular antigens; and the like. Examplesof hematological malignancy antigens include, e.g., CD19 (as expressedin e.g., B-cells), CD20 (as expressed in e.g., B-cells), CD22 (asexpressed in e.g., B-cells), CD30 (as expressed in e.g., B-cells), CD33(as expressed in e.g., Myeloid cells), CD70 (as expressed in e.g.,B-cell/T-cells), CD123 (as expressed in e.g., Myeloid cells), Kappa (asexpressed in e.g., B-cells), Lewis Y (as expressed in e.g., Myeloidcells), NKG2D ligands (as expressed in e.g., Myeloid cells), RORI (asexpressed in e.g., B-cells), SLAMF7/CS1 (as expressed in e.g., myelomacells, natural killer cells, T cells, and most B-cell types), CD138 (asexpressed in e.g., malignant plasma cells in multiple myelomas), CD56(as expressed in e.g., myeloma cells, neural cells, natural killercells, T cells, and trabecular osteoblasts) CD38 (as expressed in e.g.,B-cell/T-cells) and CD160 (as expressed in e.g., NK cells/T-cells), andthe like. Examples of solid tumor antigens include, e.g., B7H3 (asexpressed in e.g., Sarcoma, glioma), CAIX (as expressed in e.g.,Kidney), CD44 v6/v7 (as expressed in e.g., Cervical), CD171 (asexpressed in e.g., Neuroblastoma), CEA (as expressed in e.g., Colon),EGFRvIII (as expressed in e.g., Glioma), EGP2 (as expressed in e.g.,Carcinomas), EGP40 (as expressed in e.g., Colon), EphA2 (as expressed ine.g., Glioma, lung), ErbB2(HER2) (as expressed in e.g., Breast, lung,prostate, glioma), ErbB receptor family (as expressed in e.g., Breast,lung, prostate, glioma), ErbB3/4 (as expressed in e.g., Breast,ovarian), HLA-A1/MAGE1 (as expressed in e.g., Melanoma), HLA-A2/NY-ESO-1(as expressed in e.g., Sarcoma, melanoma), FR-a (as expressed in e.g.,Ovarian), FAP† (as expressed in e.g., Cancer associated fibroblasts),FAR (as expressed in e.g., Rhabdomyosarcoma), GD2 (as expressed in e.g.,Neuroblastoma, sarcoma, melanoma), GD3 (as expressed in e.g., Melanoma,lung cancer), HMW-MAA (as expressed in e.g., Melanoma), IL11Ra (asexpressed in e.g., Osteosarcoma), IL13Ra2 (as expressed in e.g.,Glioma), Lewis Y (as expressed in e.g., Breast/ovarian/pancreatic),Mesothelin (as expressed in e.g., Mesothelioma, breast, pancreas), Muc(as expressed in e.g., Ovarian, breast, prostate), NCAM (as expressed ine.g., Neuroblastoma, colorectal), NKG2D ligands (as expressed in e.g.,Ovarian, sacoma), PSCA (as expressed in e.g., Prostate, pancreatic),PSMA (as expressed in e.g., Prostate), TAG72 (as expressed in e.g.,Colon), VEGFR-2 (as expressed in e.g., Tumor vasculature), Axl (asexpressed in e.g., Lung cancer), Met (as expressed in e.g., Lungcancer), α5β3 (as expressed in e.g., Tumor vasculature), α5β1 (asexpressed in e.g., Tumor vasculature), TRAIL-R/TRAIL-R2 (as expressed ine.g., Solid tumors (colon, lung, pancreas) and hematologicalmalignancies), RANKL (as expressed in e.g., Prostate cancer and bonemetastases), Tenacin (as expressed in e.g., Glioma, epithelial tumors(breast, prostate)), EpCAM (as expressed in e.g., Epithelial tumors(breast, colon, lung)), CEA (as expressed in e.g., Epithelial tumors(breast, colon, lung)), gpA33 (as expressed in e.g., Colorectalcarcinoma), Mucins (as expressed in e.g., Epithelial tumors (breast,colon, lung, ovarian)), TAG-72 (as expressed in e.g., Epithelial tumors(breast, colon, lung)), EphA3 (as expressed in e.g., Lung, kidney,melanoma, glioma, hematological malignancies) and IGF1R (as expressed ine.g., Lung, breast, head and neck, prostate, thyroid, glioma). Examplesof surface and intracellular antigens include, e.g., Her2 (gene symbolERBB2), MAGE-A1 (gene symbol MAGEA1), MART-1 (gene symbol MLANA), NY-ESO(gene symbol CTAG), WTi (gene symbol WTi), MUC17 and MUC13. Examples ofother antigens include, e.g., BCMA (gene symbol TNFRSFi7), B7H6 (genesymbol NCR3LG1), CAIX (gene symbol CA9), CD123 (gene symbol I3RA), CD138(gene symbol SDC1), CD171 (gene symbol L1CAM), CD19 (gene symbol CD19),CD20 (gene symbol CD20), CD22 (gene symbol CD22), CD30 (gene symbolTNFRSF8), CD33 (gene symbol CD33), CD38 (gene symbol CD38), CD44, splicevariants incl 7 and 8 (denoted vX in literature) (gene symbol CD44),CEA, CS1 (gene symbol SLAMF7), EGFRvIII (gene symbol EGFR, vIII deletionvariant), EGP2, EGP40 (gene symbol EPCAM), Erb family member (genesymbol ERBB1, ERBB2, ERBB3, ERBB4), FAP (gene symbol FAP), fetalacetylcholine receptor (gene symbol AChR), Folate receptor alpha (genesymbol FOLR1), Folate receptor beta (gene symbol FOLR2), GD2, GD3, GPC3(gene symbol GPC3), Her2/neu (gene symbol ERBB2), IL-13Ra2 (gene symbolIL13RA2), Kappa light chain (gene symbol IGK), Lewis-Y, Mesothelin (genesymbol MSLN), Mucin-1 (gene symbol MUC1), Mucin-16 (gene symbol MUC16),NKG2D ligands, prostate specific membrane antigen (PSMA) (gene symbolFOLH1), prostate stem cell antigen (PSCA) (gene symbol PSCA), receptortyrosine kinase-like orphan receptor 1 (gene symbol RORI), andAnaplastic Lymphoma Receptor Tyrosine Kinase (gene symbol ALK). In somecases, the first synNotch polypeptide and the second synNotchpolypeptide specifically bind two different antigens.

In some cases, the intracellular domain of a chimeric Notch receptorpolypeptide of the present disclosure, when released upon binding of thefirst member of the specific binding pair to a second member of thespecific binding pair, induces production of a TANGO polypeptide in acell that expresses the chimeric Notch polypeptide.

As the intracellular domain of a chimeric Notch receptor polypeptide ofthe present disclosure, when released upon binding of the first memberof the specific binding pair to a second member of the specific bindingpair, may induce the expression of various polypeptides as describedherein, in some instances, induced expression of two or morepolypeptides may generate a logic gated circuit. Such logic gatedcircuits may include but are not limited to e.g., “AND gates”, “ORgates”, “NOT gates” and combinations thereof including e.g., higherorder gates including e.g., higher order AND gates, higher order ORgates, higher order NOT gates, higher order combined gates (i.e., gatesusing some combination of AND, OR and/or NOT gates).

“AND” gates of the present disclosure include where two or more inputsare required for propagation of a signal. For example, in someinstances, an AND gate allows signaling through two or morebinding-triggered transcriptional switches or portions thereof where twoinputs, e.g., two antigens, are required for signaling through the twoor more binding-triggered transcriptional switches or portions thereof.

“OR” gates of the present disclosure include where either of two or moreinputs may allow for the propagation of a signal. For example, in someinstances, an OR gate allows signaling through two or morebinding-triggered transcriptional switches or portions thereof where anyone input, e.g., either of two antigens, may induce the signaling outputof the two or more binding-triggered transcriptional switches orportions thereof.

“NOT” gates of the present disclosure include where an input is capableof preventing the propagation of a signal. For example, in someinstances, a NOT gate inhibits signaling through a binding-triggeredtranscriptional switch. In one embodiment, a NOT gate may include theinhibition of a binding interaction. For example, a competitiveinhibitor that prevents the binding of parts of a splitbinding-triggered transcriptional switch may serve as a NOT gate thatprevents signaling through the binding-triggered transcriptional switch.In another embodiment, a NOT gate may include functional inhibition ofan element of a circuit. For example, an inhibitor that functionallyprevents signaling through a binding-triggered transcriptional switch orthe outcome of signaling through a binding-triggered transcriptionalswitch may serve as a NOT gate of a molecular circuit as describedherein. As one example, an inhibitor domain, e.g., an inhibitory PD-1domain, may serve as a NOT gate to prevent signaling through abinding-triggered transcriptional switch, e.g., that results in cellactivation.

Multi-input gates may make use of a NOT gate in various different waysto prevent signaling through some other component of a circuit or turnoff a cellular response when and/or where a signal activating the NOTgate (e.g., a particular negative antigen) is present. For example, anAND+NOT gate may include a binding triggered switch that positivelyinfluences a particular cellular activity in the presence of a firstantigen and a binding triggered switch the negatively influences thecellular activity in the presence of a second antigen.

In one embodiment, a first binding-triggered transcriptional switchresponsive to antigen A drives expression of a CAR that is responsive toantigen X such that in the presence of antigens A and X the CAR isactive, resulting in T cell activation (see FIG. 136). The circuitfurther includes a second binding-triggered transcriptional switch that,in the presence of antigen B, represses the CAR (e.g., through aninhibitory intracellular domain (e.g., PD-1, CLTA4, CD45, etc.)preventing T cell activation (FIG. 136). Therefore, in the describedembodiment of a 3 input AND+NOT gate, only when antigens A and X but notB are present is the cellular activity of T cell activation induced.

In some instances, higher order multi-input gates include a NOT gatefunction. For example, in a circuit where activation relies uponexpression of two parts of a split transcription factor in an AND gateto induce a desired cellular activity a NOT functionality may beemployed, e.g., to repress the activity of the split transcriptionfactor.

In one embodiment, a first SynNotch responsive to antigen A induces theexpression or releases a first part of a split transcription factor inthe presence of antigen A and a second SynNotch responsive to antigen Binduces the expression or releases a second part of the splittranscription factor in the presence of antigen B such that when thefirst and second parts of the split transcription factor are presentand/or free the transcription factor activates some downstream activityincluding, e.g., the expression of a CAR responsive to antigen D (seeFIG. 137). In such an embodiment, the circuit may include a NOTfunctionality, e.g., in the form of a third SynNotch receptor responsiveto antigen C that, in the presence of antigen C, induces or releases adominant negative inhibitor of the split transcription factor. Anyconvenient dominant negative inhibitor of the transcription factor mayfind use in such a NOT functionality including but not limited to, e.g.,a part of the split transcription factor that lacks a domain requiredfor the two parts to form a functional transcription factor (e.g., theinteraction domain). Therefore, in the described embodiment of amulti-input gate with NOT functionality (FIG. 137), only when antigensA, B and D but not C are present is the cellular activity of the CAR,e.g., T cell activation, induced.

In another embodiment, a first SynNotch responsive to antigen A inducesthe expression of the first part of a split CAR responsive to antigen Xand a second SynNotch responsive to antigen B induces the expression ofthe second part of the split CAR responsive to antigen X such that whenantigens A and B are present the two parts of the split CAR form afunctional CAR responsive to antigen X which results in T cellactivation in the presence of antigen X (see FIG. 138). Such a circuitmay further include a NOT functionality in the form of a third SynNotchresponsive to antigen C such that in the presence of antigen C anintracellular inhibitory domain is expressed or released that inhibits Tcell activation from the split CAR (see FIG. 138). Therefore, in thedescribed embodiment of a four-input gate with NOT functionality (FIG.138), only when antigens A, B and X but not C are present is thecellular activity of the gate, e.g., T cell activation through a splitCAR, induced.

In some instances, a two input gated circuit may include a firstSynNotch polypeptide that, when specifically bound by its respectiveantigen, induces the expression of a part of a split CAR. In someinstances, a cell of the instant disclosure may constitutively express afirst part of the split CAR and thus, upon induced expression of thesecond part of the split CAR the split CAR becomes responsive to itsantigen when present. The necessity for the expression of the secondpart of a split CAR may in some instances be referred to as “priming”such that expression of the second part of the split CAR primes thesystem for response to the antigen to which the split CAR is responsive.

The configurations of such two antigen gated circuits may vary. In someinstances, a SynNotch responsive to a first antigen (i.e., antigen A)induces the expression of a part of the split CAR containing the antigenrecognition domain responsive to a second antigen (i.e., antigen X) (seee.g., FIG. 130). In such a configuration the split CAR does notrecognize antigen X until the cell is primed by expression of the partof the split CAR induced by the SynNotch polypeptide. Accordingly, thepresence of both antigen A and antigen X is required for T cellactivation.

In some instances, a SynNotch responsive to a first antigen (i.e.,antigen A) induces the expression of a part of a split CAR, responsiveto a second antigen (i.e., antigen X), containing one or moreintracellular components necessary for T cell activation (see e.g., FIG.131). In such a configuration the split CAR does recognize antigen Xprior to priming but, in the absence of antigen A, the binding ofantigen X is not propagated to induce T cell activation. However, whenthe part of the split CAR induced by the SynNotch polypeptide isexpressed the binding of antigen X is propagated leading to T cellactivation. Accordingly, the presence of both antigen A and antigen X isrequired for T cell activation.

In some instances, a three input AND gate may include two SynNotchpolypeptides that, when specifically bound by their respective first andsecond antigens, induce expression of a third antigen-responsivepolypeptide that becomes activated upon binding of a third antigen. Forexample, in one embodiment a three input AND gate may include a firstSynNotch polypeptide responsive to a first antigen and a second SynNotchpolypeptide responsive to a second antigen wherein the first and secondSynNotch polypeptides induce expression of first and second parts of asplit CAR that is responsive to a third antigen. Thus, in the presenceof the first and second antigens (i.e., antigens A and B) the first andsecond parts of the split CAR are expressed and, in the presence of thethird antigen (i.e., antigen C), the split CAR activates the T cell inwhich it is expressed (see FIG. 132). A further schematic of a threeantigen gating system where two SynNotch polypeptides recognizingantigen A and antigen B induce the expression of parts of a split CARthat recognizes antigen X is depicted in FIG. 133. In such a system,antigens A, B and X are required for T cell activation.

In some instances, a four input AND gate may include three SynNotchpolypeptides that, when specifically bound by their respective first,second and third antigens, induce expression, directly or indirectly, ofa fourth antigen-responsive polypeptide that becomes activated uponbinding of a fourth antigen. For example, in one embodiment a four inputAND gate may include a first SynNotch polypeptide responsive to a firstantigen, a second SynNotch polypeptide responsive to a second antigenand a third SynNotch polypeptide responsive to a third antigen, whereinthe first SynNotch polypeptide induces expression of a first part of asplit CAR that is responsive to a fourth antigen and the second andthird SynNotch polypeptides induce expression of first and second partsof a transcription factor that, when both parts are present, induceexpression of the second part of the split CAR that is responsive to thefourth antigen. This, in the presence of the first, second and thirdantigens (i.e., antigens A, B and C) the first and second parts of thesplit CAR are expressed and, in the presence of the fourth antigen(i.e., antigen D), the split CAR activates the T cell in which it isexpressed (see FIG. 134).

In some instances, a five input AND gate may include four SynNotchpolypeptides that, when specifically bound by their respective first,second, third and fourth antigens, induce expression of a fifthantigen-responsive polypeptide that becomes activated upon binding of afifth antigen. For example, in one embodiment a five input AND gate mayinclude first and second SynNotch polypeptides, responsive to first andsecond antigens, that induce expression of first and second parts of afirst transcription factor that, when both parts are present, inducesexpression of a first part of a split CAR and third and fourth SynNotchpolypeptides, responsive to third and fourth antigens, that induceexpression of first and second parts of a second transcription factorthat, when both parts are present, induces expression of the second partof a split CAR that is responsive to a fifth antigen. Thus, in thepresence of the first, second, third and fourth antigens (i.e., antigensA, B, C and D) the first and second parts of the split CAR are expressedand, in the presence of the fifth antigen (i.e., antigen E), the splitCAR activates the T cell in which it is expressed (see FIG. 135).

Where split transcription factors are utilized, e.g., as in logic gatedSynNotch circuits, the transcription factor or portion thereof may beexpressed within a cell from an expression cassette separate from otherexpression cassettes of the system, e.g., expression cassettescontaining sequence encoding a SynNotch or portion thereof, sequenceencoding a CAR or portion thereof, etc. Where a transcription factor orportion thereof, e.g., a portion of a split transcription factor, iscontained in an expression cassette separate from other components ofthe circuit the transcription factor expression cassette may containonly the sequence encoding the transcription factor or portion thereofand sequence elements necessary for its expression including e.g., apromoter, an enhancer, etc. A separate transcription factor or portionthereof may or may not contain further elements that do not materiallyaffect the expression or function of the transcription including, .e.g.,sequence encoding a reporter, sequence encoding a tag, etc.

In other instances, where a split transcription factor is utilized,e.g., as in logic gated SynNotch circuit, the transcription factor orportion thereof may be expressed within a cell from an expressioncassette shared with other components of the system, e.g., an expressioncassette encoding a SynNotch or portion thereof, an expression cassetteencoding a CAR or portion thereof, etc. Sequence encoding atranscription factor or portion thereof present on an expressioncassette shared with other components of the system may be independentlycontrolled, i.e., contain expression control elements (i.e., promoters,enhancers, etc.) separate from the other system elements of thecassette, or may be simultaneously controlled with the other systemelements of the expression cassette, i.e., the transcription factor andone or more of the other system elements are controlled from the sameexpression control elements. Where sequence encoding a transcriptionfactor or portion thereof is simultaneously controlled with the othersystem elements of the expression cassette the transcription factor orportion thereof may be encoded to be attached to one or more of theother system elements.

In certain embodiments, a nucleic acid encoding a binding-triggeredtranscriptional switch is configured to encode a portion of splittranscription factor operably linked to one or more domains of thebinding-triggered transcriptional switch such that, upon activation ofthe binding-triggered transcriptional switch, the portion of the splittranscription factor is released and available to complex with one ormore other portions of the split transcription factor to form afunctional transcription factor.

Accordingly, activation of one or more binding-triggered transcriptionalswitches may induce expression of portions of split transcriptionfactors resulting in heterodimerization and/or complex formation of thesplit transcription factor portions resulting in formation of afunctional transcription factor. Alternatively, activation of one ormore binding-triggered transcriptional switches may result in release ofsplit transcription factor portions from the one or morebinding-triggered transcriptional switches resulting inheterodimerization and/or complex formation of the split transcriptionfactor portions resulting in formation of a functional transcriptionfactor. In addition, induction and release of split transcription factorportions may be combined, e.g., where activation of one or morebinding-triggered transcriptional switches may induce expression ofportions of split transcription factors and release of splittranscription factor portions from the one or more binding-triggeredtranscriptional switches resulting in heterodimerization and/or complexformation of the split transcription factor portions resulting information of a functional transcription factor.

Logic gated systems of the instant disclosure are not limited to thosespecifically described and may include alternative configurations and/orhigher order gates as compared to those described. For example, in someinstances a logic gated system of the instant disclosure may be a twoinput gate, a three input gate, a four input gate, a five input gate, asix input gate, a seven input gate, an eight input gate, a nine inputgate, a ten input gate or greater. Furthermore, the components of logicgated systems of the instant disclosure are not limited to SynNotch forthe induction of expression of further circuit components and mayinclude e.g., other binding-triggered transcriptional switches includingbut not limited to e.g., those described herein. In addition, while theforegoing examples have been described, for simplicity, in terms ofinduced expression of the parts of a split CAR, other split moleculesincluding but not limited to e.g., split SynNotch polypeptides, may finduse in logic gated circuits of the instant disclosure.

The present disclosure provides a method of modulating the activity of atarget cell, the method comprising: a) expressing in the target cell afirst synNotch polypeptide of the present disclosure, where the firstsynNotch polypeptide comprises an antigen-binding domain (e.g., an scFv,a nanobody, etc.) that binds a first epitope on a soluble adaptermolecule (e.g., an antigen); b) contacting the target cell with: i) asecond cell that expresses a second synNotch polypeptide of the presentdisclosure, where the second synNotch polypeptide comprising anantigen-binding domain (e.g., an scFv, a nanobody, etc.) that binds asecond epitope on the soluble adapter molecule (e.g., an antigen); andii) the soluble adapter molecule (e.g. an antigen), wherein binding ofthe antigen-binding domain of the first synNotch polypeptide and theantigen-binding domain of the second synNotch polypeptide to the solubleadapter molecule (e.g., an antigen) induces cleavage of theintracellular domain of the first synNotch polypeptide, therebyreleasing the intracellular domain, wherein the released intracellulardomain modulates an activity of the target cell. The activity of thetarget cell can be selected from the group consisting of: expression ofa gene product of the cell, proliferation of the cell, apoptosis of thecell, non-apoptotic death of the cell, differentiation of the cell,dedifferentiation of the cell, migration of the cell, secretion of amolecule from the cell and cellular adhesion of the cell. The adaptormolecule can be a cancer-associated antigen, a pathogen-associatedantigen, an antibody, and the like.

Additional Sequences

A chimeric Notch receptor polypeptide of the present disclosure canfurther include one or more additional polypeptides, where suitableadditional polypeptides include, but are not limited to, a signalsequence; an epitope tag; an affinity domain; a nuclear localizationsignal (NLS); and a polypeptide that produces a detectable signal.

Signal Sequences

Signal sequences that are suitable for use in a chimeric Notch receptorpolypeptide of the present disclosure include any eukaryotic signalsequence, including a naturally-occurring signal sequence, a synthetic(e.g., man-made) signal sequence, etc.

Epitope Tag

Suitable epitope tags include, but are not limited to, hemagglutinin(HA; e.g., YPYDVPDYA (SEQ ID NO:73); FLAG (e.g., DYKDDDDK (SEQ IDNO:74); c-myc (e.g., EQKLISEEDL; SEQ ID NO:75), and the like.

Affinity Domain

Affinity domains include peptide sequences that can interact with abinding partner, e.g., such as one immobilized on a solid support,useful for identification or purification. Multiple consecutive singleamino acids, such as histidine, when fused to a chimeric Notch receptorpolypeptide of the present disclosure, may be used for one-steppurification of the recombinant chimeric polypeptide by high affinitybinding to a resin column, such as nickel sepharose. Exemplary affinitydomains include His5 (HHHHH) (SEQ ID NO:76), HisX6 (HHHHHH) (SEQ IDNO:77), C-myc (EQKLISEEDL) (SEQ ID NO:75), Flag (DYKDDDDK) (SEQ IDNO:74), StrepTag (WSHPQFEK) (SEQ ID NO:78), hemagglutinin, e.g., HA Tag(YPYDVPDYA) (SEQ ID NO:73), GST, thioredoxin, cellulose binding domain,RYIRS (SEQ ID NO:79), Phe-His-His-Thr (SEQ ID NO:80), chitin bindingdomain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag,WEAAAREACCRECCARA (SEQ ID NO:81), metal binding domains, e.g., zincbinding domains or calcium binding domains such as those fromcalcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B,myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin,hippocalcin, frequenin, caltractin, calpain large-subunit, S100proteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin,inteins, biotin, streptavidin, MyoD, Id, leucine zipper sequences, andmaltose binding protein.

Nuclear Localization Sequences

Suitable nuclear localization signals (“NLS”; also referred to herein as“nuclear localization sequences”) include, e.g., PKKKRKV (SEQ ID NO:82);KRPAATKKAGQAKKKK (SEQ ID NO:83); MVPKKKRK (SEQ ID NO:84);MAPKKKRKVGIHGVPAA (SEQ ID NO:85); and the like. An NLS can be present atthe N-terminus of a chimeric Notch receptor polypeptide of the presentdisclosure; near the N-terminus of a chimeric Notch receptor polypeptideof the present disclosure (e.g., within 5 amino acids, within 10 aminoacids, or within 20 amino acids of the N-terminus); at the C-terminus ofa chimeric Notch receptor polypeptide of the present disclosure; nearthe C-terminus of a chimeric Notch receptor polypeptide of the presentdisclosure (e.g., within 5 amino acids, within 10 amino acids, or within20 amino acids of the C-terminus); or internally within a chimeric Notchreceptor polypeptide of the present disclosure.

Detectable Signal-Producing Polypeptides

Suitable detectable signal-producing proteins include, e.g., fluorescentproteins; enzymes that catalyze a reaction that generates a detectablesignal as a product; and the like.

Suitable fluorescent proteins include, but are not limited to, greenfluorescent protein (GFP) or variants thereof, blue fluorescent variantof GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescentvariant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhancedYFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine,GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP),destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet,mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2,t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP,Kaede protein and kindling protein, Phycobiliproteins andPhycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrinand Allophycocyanin. Other examples of fluorescent proteins includemHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry,mCherry, mGrape1, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat.Methods 2:905-909), and the like. Any of a variety of fluorescent andcolored proteins from Anthozoan species, as described in, e.g., Matz etal. (1999) Nature Biotechnol. 17:969-973, is suitable for use.

Suitable enzymes include, but are not limited to, horse radishperoxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL),glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase,β-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase,glucose oxidase (GO), and the like.

Examples of Second Members of Specific Binding Pairs

As noted above, binding of the first member of the specific binding pairof a chimeric Notch polypeptide of the present disclosure to a secondmember of the specific binding pair induces cleavage of the Notchreceptor polypeptide at the one or more ligand-inducible proteolyticcleavage sites, thereby releasing the intracellular domain. As notedabove, the second member of the specific binding pair can be any of avariety of molecules. In some cases, a chimeric Notch polypeptidespecifically binds an antigen; e.g., the second member of the specificbinding pair is an antigen. Examples of such antigens include, e.g.,tumor antigens; cancer cell-associated antigens; hematologicalmalignancy antigens; solid tumor antigens; cell surface antigens (e.g.,cell surface antigens targeted by a T cell receptor (TCR); intracellularantigens; and the like. Examples of hematological malignancy antigensinclude, e.g., CD19 (as expressed in e.g., B-cells), CD20 (as expressedin e.g., B-cells), CD22 (as expressed in e.g., B-cells), CD30 (asexpressed in e.g., B-cells), CD33 (as expressed in e.g., Myeloid cells),CD70 (as expressed in e.g., B-cell/T-cells), CD123 (as expressed ine.g., Myeloid cells), Kappa (as expressed in e.g., B-cells), Lewis Y (asexpressed in e.g., Myeloid cells), NKG2D ligands (as expressed in e.g.,Myeloid cells), RORI (as expressed in e.g., B-cells), SLAMF7/CS1 (asexpressed in e.g., myeloma cells, natural killer cells, T cells, andmost B-cell types), CD138 (as expressed in e.g., malignant plasma cellsin multiple myelomas), CD56 (as expressed in e.g., myeloma cells, neuralcells, natural killer cells, T cells, and trabecular osteoblasts) CD38(as expressed in e.g., B-cell/T-cells) and CD160 (as expressed in e.g.,NK cells/T-cells), and the like. Examples of solid tumor antigensinclude, e.g., B7H3 (as expressed in e.g., Sarcoma, glioma), CAIX (asexpressed in e.g., Kidney), CD44 v6/v7 (as expressed in e.g., Cervical),CD171 (as expressed in e.g., Neuroblastoma), CEA (as expressed in e.g.,Colon), EGFRvIII (as expressed in e.g., Glioma), EGP2 (as expressed ine.g., Carcinomas), EGP40 (as expressed in e.g., Colon), EphA2 (asexpressed in e.g., Glioma, lung), ErbB2(HER2) (as expressed in e.g.,Breast, lung, prostate, glioma), ErbB receptor family (as expressed ine.g., Breast, lung, prostate, glioma), ErbB3/4 (as expressed in e.g.,Breast, ovarian), HLA-A1/MAGE1 (as expressed in e.g., Melanoma),HLA-A2/NY-ESO-1 (as expressed in e.g., Sarcoma, melanoma), FR-a (asexpressed in e.g., Ovarian), FAP† (as expressed in e.g., Cancerassociated fibroblasts), FAR (as expressed in e.g., Rhabdomyosarcoma),GD2 (as expressed in e.g., Neuroblastoma, sarcoma, melanoma), GD3 (asexpressed in e.g., Melanoma, lung cancer), HMW-MAA (as expressed ine.g., Melanoma), IL11Ra (as expressed in e.g., Osteosarcoma), IL13Ra2(as expressed in e.g., Glioma), Lewis Y (as expressed in e.g.,Breast/ovarian/pancreatic), Mesothelin (as expressed in e.g.,Mesothelioma, breast, pancreas), Muc (as expressed in e.g., Ovarian,breast, prostate), NCAM (as expressed in e.g., Neuroblastoma,colorectal), NKG2D ligands (as expressed in e.g., Ovarian, sacoma), PSCA(as expressed in e.g., Prostate, pancreatic), PSMA (as expressed ine.g., Prostate), TAG72 (as expressed in e.g., Colon), VEGFR-2 (asexpressed in e.g., Tumor vasculature), Axl (as expressed in e.g., Lungcancer), Met (as expressed in e.g., Lung cancer), α5β3 (as expressed ine.g., Tumor vasculature), α5β1 (as expressed in e.g., Tumorvasculature), TRAIL-R/TRAIL-R2 (as expressed in e.g., Solid tumors(colon, lung, pancreas) and hematological malignancies), RANKL (asexpressed in e.g., Prostate cancer and bone metastases), Tenacin (asexpressed in e.g., Glioma, epithelial tumors (breast, prostate)), EpCAM(as expressed in e.g., Epithelial tumors (breast, colon, lung)), CEA (asexpressed in e.g., Epithelial tumors (breast, colon, lung)), gpA33 (asexpressed in e.g., Colorectal carcinoma), Mucins (as expressed in e.g.,Epithelial tumors (breast, colon, lung, ovarian)), TAG-72 (as expressedin e.g., Epithelial tumors (breast, colon, lung)), EphA3 (as expressedin e.g., Lung, kidney, melanoma, glioma, hematological malignancies) andIGF1R (as expressed in e.g., Lung, breast, head and neck, prostate,thyroid, glioma). Examples of surface and intracellular antigensinclude, e.g., Her2 (gene symbol ERBB2), MAGE-A1 (gene symbol MAGEA1),MART-1 (gene symbol MLANA), NY-ESO (gene symbol CTAG1), WT1 (gene symbolWT1), MUC17 and MUC13. Examples of other antigens include, e.g., BCMA(gene symbol TNFRSFi7), B7H6 (gene symbol NCR3LG1), CAIX (gene symbolCA9), CD123 (gene symbol IL3RA), CD138 (gene symbol SDCI), CD171 (genesymbol LCAM), CD19 (gene symbol CD19), CD20 (gene symbol CD20), CD22(gene symbol CD22), CD30 (gene symbol TNFRSF8), CD33 (gene symbol CD33),CD38 (gene symbol CD38), CD44, splice variants incl 7 and 8 (denoted vXin literature) (gene symbol CD44), CEA, CS1 (gene symbol SLAMF7),EGFRvIII (gene symbol EGFR, vIII deletion variant), EGP2, EGP40 (genesymbol EPCAM), Erb family member (gene symbol ERBB1, ERBB2, ERBB3,ERBB4), FAP (gene symbol FAP), fetal acetylcholine receptor (gene symbolAChR), Folate receptor alpha (gene symbol FOLR1), Folate receptor beta(gene symbol FOLR2), GD2, GD3, GPC3 (gene symbol GPC3), Her2/neu (genesymbol ERBB2), IL-13Ra2 (gene symbol IL13RA2), Kappa light chain (genesymbol IGK), Lewis-Y, Mesothelin (gene symbol MSLN), Mucin-1 (genesymbol MUC1), Mucin-16 (gene symbol MUC16), NKG2D ligands, prostatespecific membrane antigen (PSMA) (gene symbol FOLH1), prostate stem cellantigen (PSCA) (gene symbol PSCA), receptor tyrosine kinase-like orphanreceptor 1 (gene symbol RORI), and Anaplastic Lymphoma Receptor TyrosineKinase (gene symbol ALK).

Exemplary Embodiments

The following are non-limiting examples of a chimeric Notch receptorpolypeptide of the present disclosure.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises a Notch receptor polypeptide thatcomprises, in order from N-terminus to C-terminus: i) Lin Notch RepeatsA-C (an LNR segment); ii) a heterodimerization domain (an HD-N segmentand an HD-C segment); iii) a TM domain; and comprises an S1 proteolyticcleavage site, an S2 proteolytic cleavage site, and an S3 proteolyticcleavage site. An example of such a Notch receptor polypeptide isdepicted in FIG. 16A. In FIG. 16A, Lin Notch Repeats A-C (an LNRsegment) have the following amino acid sequence:PPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECE WDGLDC (SEQ IDNO:5); the heterodimerization domain (an HD-N segment and an HD-Csegment) has the following amino acid sequence:AAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLP (SEQ ID NO:6), where the S1proteolytic cleavage site includes the sequence RQRR (SEQ ID NO:86), andthe S2 proteolytic cleavage site includes the sequence AV; and the TMdomain has the following amino acid sequence: HLMYVAAAAFVLLFFVGCGVLLS(SEQ ID NO:7), where the S3 proteolytic cleavage site includes thesequence VLLS (SEQ ID NO:87).

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises a Notch receptor polypeptide thatcomprises, in order from N-terminus to C-terminus: i) an EGF repeat; ii)Lin Notch Repeats A-C (an LNR segment); iii) a heterodimerization domain(an HD-N segment and an HD-C segment); iv) a TM domain; and comprises anS1 proteolytic cleavage site, an S2 proteolytic cleavage site, and an S3proteolytic cleavage site. An example of such a Notch receptorpolypeptide is depicted in FIG. 16B. In FIG. 16B, the EGF repeat has thefollowing amino acid sequence: PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCH(SEQ ID NO:8); and the LNR segment, the heterodimerization domain, theTM domain, the S1 proteolytic cleavage site, the S2 proteolytic cleavagesite, and the S3 proteolytic cleavage site are as depicted in FIG. 16A.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises a Notch receptor polypeptide thatcomprises the following amino acid sequence:IPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRRQLCIQKL (SEQ ID NO:4);where the TM domain is underlined; where the Notch receptor polypeptidecomprises an S2 proteolytic cleavage site and an S3 proteolytic cleavagesite; where the Notch receptor polypeptide has a length of 56 aminoacids.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an Fc receptor FcγIIIa (CD16A); b) a Notch receptorpolypeptide comprising: i) an LNR segment; ii) a heterodimerizationdomain (an HD-N segment and an HD-C segment); and iii) a TM domain,where the Notch receptor polypeptide comprises one or moreligand-inducible proteolytic cleavage sites; and c) an intracellulardomain, where the intracellular domain is a transcriptional activator.In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an Fc receptor FcγIIIa (CD16A); a Notch receptorpolypeptide comprising: i) an LNR segment; ii) a heterodimerizationdomain (an HD-N segment and an HD-C segment); and iii) a TM domain,where the Notch receptor polypeptide comprises one or moreligand-inducible proteolytic cleavage sites; and c) an intracellulardomain, where the intracellular domain is a tTA transcription factor. Anexample of such a chimeric Notch receptor polypeptide is depicted inFIG. 16C. The locations of S1, S2, and S3 cleavage sites are depicted inFIG. 16A. In FIG. 16C, the CD16A has the following amino acid sequence:MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPG (SEQ ID NO:88); the Notch receptorpolypeptide has the amino acid sequence depicted in FIG. 16A; and thetTA transcription factor has the following amino acid sequence:

(SEQ ID NO: 69) MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) a cell surface antigen; b) a Notch receptor polypeptidecomprising: i) an LNR segment; ii) a heterodimerization domain (an HD-Nsegment and an HD-C segment); and iii) a TM domain, where the Notchreceptor polypeptide comprises one or more ligand-inducible proteolyticcleavage sites; and c) an intracellular domain, where the intracellulardomain is a transcriptional activator. In one non-limiting embodiment, achimeric Notch receptor polypeptide of the present disclosure comprises,in order from N-terminus to C-terminus: a) a CD19 polypeptide; b) aNotch receptor polypeptide comprising: i) an LNR segment; ii) aheterodimerization domain (an HD-N segment and an HD-C segment); andiii) a TM domain where the Notch receptor polypeptide comprises one ormore ligand-inducible proteolytic cleavage sites; and c) anintracellular domain, where the intracellular domain is a tTAtranscription factor. An example of such a chimeric Notch receptorpolypeptide is depicted in FIG. 17A. In FIG. 17A, the CD19 polypeptidehas the following amino acid sequence:RPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWK (SEQ ID NO:89); the Notchreceptor polypeptide includes the amino acid sequence depicted in FIG.16A; and the tTA transcription factor has the amino acid sequencedepicted in FIG. 16C.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an antibody; b) a Notch receptor polypeptide comprising:i) an LNR segment; ii) a heterodimerization domain (an HD-N segment andan HD-C segment); and iii) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa transcriptional activator. In one non-limiting embodiment, a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) an antibody specific for a cellsurface antigen; b) a Notch receptor polypeptide comprising: i) an LNRsegment; ii) a heterodimerization domain (an HD-N segment and an HD-Csegment); and iii) a TM domain, where the Notch receptor polypeptidecomprises one or more ligand-inducible proteolytic cleavage sites; andc) an intracellular domain, where the intracellular domain is atranscriptional activator. In one non-limiting embodiment, a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) an anti-CD19 scFv; b) a Notch receptorpolypeptide comprising: i) an LNR segment; ii) a heterodimerizationdomain (an HD-N segment and an HD-C segment); and iii) a TM domain wherethe Notch receptor polypeptide comprises one or more ligand-inducibleproteolytic cleavage sites; and c) an intracellular domain, where theintracellular domain is a tTA transcription factor. An example of such achimeric Notch receptor polypeptide is depicted in FIG. 17B. In FIG.17B, the anti-CD19 scFv has the following amino acid sequence:DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTS VTVSS (SEQ IDNO:90); the Notch receptor polypeptide includes the amino acid sequencedepicted in FIG. 16A; and the tTA transcription factor has the aminoacid sequence depicted in FIG. 16C.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an antibody; b) a Notch receptor polypeptide comprising:i) an EGF repeat; ii) an LNR segment; iii) a heterodimerization domain(an HD-N segment and an HD-C segment); and iv) a TM domain, where theNotch receptor polypeptide comprises one or more ligand-inducibleproteolytic cleavage sites; and c) an intracellular domain, where theintracellular domain is a transcriptional activator. In one non-limitingembodiment, a chimeric Notch receptor polypeptide of the presentdisclosure comprises, in order from N-terminus to C-terminus: a) anantibody specific for a cell surface antigen; b) a Notch receptorpolypeptide comprising: i) an EGF repeat; ii) an LNR segment; iii) aheterodimerization domain (an HD-N segment and an HD-C segment); and iv)a TM domain, where the Notch receptor polypeptide comprises one or moreligand-inducible proteolytic cleavage sites; and c) an intracellulardomain, where the intracellular domain is a transcriptional activator.In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an anti-CD19 scFv; b) a Notch receptor polypeptidecomprising: i) an EGF repeat; ii) an LNR segment; iii) aheterodimerization domain (an HD-N segment and an HD-C segment); and iv)a TM domain, where the Notch receptor polypeptide comprises one or moreligand-inducible proteolytic cleavage sites; and c) an intracellulardomain, where the intracellular domain is a tTA transcription factor. Anexample of such a chimeric Notch receptor polypeptide is depicted inFIG. 17C. In FIG. 17C, the anti-CD19 scFv has the following amino acidsequence: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTS VTVSS (SEQ IDNO:90); the Notch receptor polypeptide includes the amino acid sequencedepicted in FIG. 16B; and the tTA transcription factor has the aminoacid sequence depicted in FIG. 16C.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an antibody; b) a Notch receptor polypeptide comprising:i) an EGF repeat; ii) an LNR segment; iii) a heterodimerization domain(an HD-N segment and an HD-C segment); and iv) a TM domain, where theNotch receptor polypeptide comprises one or more ligand-inducibleproteolytic cleavage sites; and c) an intracellular domain, where theintracellular domain is a transcriptional activator. In one non-limitingembodiment, a chimeric Notch receptor polypeptide of the presentdisclosure comprises, in order from N-terminus to C-terminus: a) anantibody specific for a cell surface antigen, e.g., a cell surfaceantigen present on the surface of a cancer cell (e.g., a cancer-specificantigen); b) a Notch receptor polypeptide comprising: i) an EGF repeat;ii) an LNR segment; iii) a heterodimerization domain (an HD-N segmentand an HD-C segment); and iv) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa transcriptional activator. In one non-limiting embodiment, a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) an anti-mesothelin scFv; b) a Notchreceptor polypeptide comprising: i) an EGF repeat; ii) an LNR segment;iii) a heterodimerization domain (an HD-N segment and an HD-C segment);and iv) a TM domain, where the Notch receptor polypeptide comprises oneor more ligand-inducible proteolytic cleavage sites; and c) anintracellular domain, where the intracellular domain is a tTAtranscription factor. An example of such a chimeric Notch receptorpolypeptide is depicted in FIG. 18. In FIG. 18, the anti-mesothelin scFvhas the following amino acid sequence:GSQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSSGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTYG AGTKLEIKAS(SEQ ID NO:91); the Notch receptor polypeptide includes the amino acidsequence depicted in FIG. 16B; and the tTA transcription factor has theamino acid sequence depicted in FIG. 16C.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an antibody; b) a Notch receptor polypeptide comprising:i) an LNR segment; ii) a heterodimerization domain (an HD-N segment andan HD-C segment); and iii) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa transcriptional activator. In one non-limiting embodiment, a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) an antibody specific for atranscription factor; b) a Notch receptor polypeptide comprising: i) anLNR segment; ii) a heterodimerization domain (an HD-N segment and anHD-C segment); and iii) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa transcriptional activator. In one non-limiting embodiment, a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) an anti-myc scFv; b) a Notch receptorpolypeptide comprising: i) an LNR segment; ii) a heterodimerizationdomain (an HD-N segment and an HD-C segment); and iii) a TM domain,where the Notch receptor polypeptide comprises one or moreligand-inducible proteolytic cleavage sites; and c) an intracellulardomain, where the intracellular domain is a tTA transcription factor.Examples of such a chimeric Notch receptor polypeptide are depicted inFIGS. 19A and 19B. In FIGS. 19A and 19B, the anti-Myc scFv has thefollowing amino acid sequence:GSQVQLQQQVQLQESGGDLVKPGGSLKLSCAASGFTFSHYGMSWVRQTPDKRLEWVATIGSRGTYTHYPDSVKGRFTISRDNDKNALYLQMNSLKSEDTAMYYCARRSEFYYYGNTYYYSAMDYWGQGASVTVSSGGGGSGGGGSGGGGSDIVLTQSPAFLAVSLGQRATISCRASESVDNYGFSFMNWFQQKPGQPPKLLIYAISNRGSGVPARFSGSGSGTDFSLNIHPVEEDDPAMYFCQQTKEVPWTFGGGTKLEIK (SEQ ID NO:92); the Notch receptorpolypeptide includes the amino acid sequence depicted in FIG. 16A; andthe tTA transcription factor has the amino acid sequence depicted inFIG. 16C.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) a nanobody; b) a Notch receptor polypeptide comprising:i) an LNR segment; ii) a heterodimerization domain (an HD-N segment andan HD-C segment); and iii) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa transcriptional activator. In one non-limiting embodiment, a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) a LaG 9 nanobody; b) a Notch receptorpolypeptide comprising: i) an LNR segment; ii) a heterodimerizationdomain (an HD-N segment and an HD-C segment); and iii) a TM domain,where the Notch receptor polypeptide comprises one or moreligand-inducible proteolytic cleavage sites; and c) an intracellulardomain, where the intracellular domain is a tTA transcription factor. Anexample of such a chimeric Notch receptor polypeptide is depicted inFIG. 20A. In FIG. 20A, the LaG 9 nanobody has the following amino acidsequence: MADVQLVESGGGLVQAGGSLRLSCAASGRTFSTSAMGWFRQAPGKEREFVARITWSAGYTAYSDSVKGRFTISRDKAKNTVYLQMNSLKPEDTAVYYCASRSAGYSSSLTRREDY AYWGQGTQVTVS(SEQ ID NO:93); the Notch receptor polypeptide includes the amino acidsequence depicted in FIG. 16A; and the tTA transcription factor has theamino acid sequence depicted in FIG. 16C.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) a nanobody; b) a Notch receptor polypeptide comprising:i) an LNR segment; ii) a heterodimerization domain (an HD-N segment andan HD-C segment); and iii) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa transcriptional activator. In one non-limiting embodiment, a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) a LaG 50 nanobody; b) a Notch receptorpolypeptide comprising: i) an LNR segment; ii) a heterodimerizationdomain (an HD-N segment and an HD-C segment); and iii) a TM domain,where the Notch receptor polypeptide comprises one or moreligand-inducible proteolytic cleavage sites; and c) an intracellulardomain, where the intracellular domain is a tTA transcription factor. Anexample of such a chimeric Notch receptor polypeptide is depicted inFIG. 20B. In FIG. 20B, the LaG 50 nanobody has the following amino acidsequence: MADVQLVESGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFVAGISRSAGSAVHADSVKGRFTISRDNTKNTLYLQMNSLKAEDTAVYYCAVRTSGFFGSIPRTGTAFD YWGQGTQVTVS(SEQ ID NO:94); the Notch receptor polypeptide includes the amino acidsequence depicted in FIG. 16A; and the tTA transcription factor has theamino acid sequence depicted in FIG. 16C.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) a nanobody; b) a Notch receptor polypeptide comprising:i) an LNR segment; ii) a heterodimerization domain (an HD-N segment andan HD-C segment); and iii) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa transcriptional activator. In one non-limiting embodiment, a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) a LaG 18 nanobody; b) a Notch receptorpolypeptide comprising: i) an LNR segment; ii) a heterodimerizationdomain (an HD-N segment and an HD-C segment); and iii) a TM domain,where the Notch receptor polypeptide comprises one or moreligand-inducible proteolytic cleavage sites; and c) an intracellulardomain, where the intracellular domain is a tTA transcription factor. Anexample of such a chimeric Notch receptor polypeptide is depicted inFIG. 20C. In FIG. 20C, the LaG 18 nanobody has the following amino acidsequence: MAQVQLVESGGGLVQTGGSLKLSCTASVRTLSYYHVGWFRQAPGKEREFVAGIHRSGESTFYADSVKGRFTISRDNAKNTVHLQMNSLKPEDTAVYYCAQRVRGFFGPLRSTPSWY DYWGQGTQVTVS(SEQ ID NO:95); the Notch receptor polypeptide includes the amino acidsequence depicted in FIG. 16A; and the tTA transcription factor has theamino acid sequence depicted in FIG. 16C.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) a nanobody; b) a Notch receptor polypeptide comprising:i) an LNR segment; ii) a heterodimerization domain (an HD-N segment andan HD-C segment); and iii) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa transcriptional activator. In one non-limiting embodiment, a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) a LaG 16/LaG 2 nanobody; b) a Notchreceptor polypeptide comprising: i) an LNR segment; ii) aheterodimerization domain (an HD-N segment and an HD-C segment); andiii) a TM domain, where the Notch receptor polypeptide comprises one ormore ligand-inducible proteolytic cleavage sites; and c) anintracellular domain, where the intracellular domain is a tTAtranscription factor. An example of such a chimeric Notch receptorpolypeptide is depicted in FIG. 20D. In FIG. 20D, the LaG 16/LaG 2nanobody has the following amino acid sequence:MAQVQLVESGGRLVQAGDSLRLSCAASGRTFSTSAMAWFRQAPGREREFVAAITWTVGNTILGDSVKGRFTISRDRAKNTVDLQMDNLEPEDTAVYYCSARSRGYVLSVLRSVDSYDYWGQGTQVTVSGGGGSGGGGSGGGGSMAQVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFVAAISWTGVSTYYADSVKGRFTISRDNDKNTVYVQMNSLIPEDTAIYYCAAVRARSFSDTYSRVNEYDYWGQGTQVTV (SEQ ID NO:96); the Notchreceptor polypeptide includes the amino acid sequence depicted in FIG.16A; and the tTA transcription factor has the amino acid sequencedepicted in FIG. 16C.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an apoptosis regulatory protein; b) a Notch receptorpolypeptide comprising: i) an EGF repeat; ii) an LNR segment; iii) aheterodimerization domain (an HD-N segment and an HD-C segment); and iv)a TM domain, where the Notch receptor polypeptide comprises one or moreligand-inducible proteolytic cleavage sites; and c) an intracellulardomain, where the intracellular domain is a transcriptional activator.In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) a programmed cell death protein 1 (PD-1) extracellulardomain; b) a Notch receptor polypeptide comprising: i) an EGF repeat;ii) an LNR segment; iii) a heterodimerization domain (an HD-N segmentand an HD-C segment); and iv) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa tTA transcription factor. An example of such a chimeric Notch receptorpolypeptide is depicted in FIG. 21. In FIG. 21, the PD1 extracellulardomain has the following amino acid sequence:MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQEQKLISEEDL (SEQ IDNO:97); the Notch receptor polypeptide includes the amino acid sequencedepicted in FIG. 16B; and the tTA transcription factor has the aminoacid sequence depicted in FIG. 16C.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an antibody; b) a Notch receptor polypeptide comprising:i) an LNR segment; ii) a heterodimerization domain (an HD-N segment andan HD-C segment); and iii) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa transcriptional activator. In one non-limiting embodiment, a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) an antibody specific for a cellsurface antigen; b) a Notch receptor polypeptide comprising: i) an LNRsegment; ii) a heterodimerization domain (an HD-N segment and an HD-Csegment); and iii) a TM domain, where the Notch receptor polypeptidecomprises one or more ligand-inducible proteolytic cleavage sites; andc) an intracellular domain, where the intracellular domain is atranscriptional activator. In one non-limiting embodiment, a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) an anti-CD19 scFv; b) a Notch receptorpolypeptide comprising: i) an LNR segment; ii) a heterodimerizationdomain (an HD-N segment and an HD-C segment); and iii) a TM domain wherethe Notch receptor polypeptide comprises one or more ligand-inducibleproteolytic cleavage sites; and c) an intracellular domain, where theintracellular domain is Gal4-VP64 transcriptional activator. An exampleof such a chimeric Notch receptor polypeptide is depicted in FIG. 22. InFIG. 22, the anti-CD19 scFv has the following amino acid sequence:DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTS VTVSS (SEQ IDNO:90); the Notch receptor polypeptide includes the amino acid sequencedepicted in FIG. 16A; and the Gal4-VP64 transcriptional activator hasthe following amino acid sequence:

(SEQ ID NO: 70) MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDA LDDFDLDMLGS.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an antibody; b) a Notch receptor polypeptide comprising:i) an LNR segment; ii) a heterodimerization domain (an HD-N segment andan HD-C segment); and iii) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa DNA binding polypeptide. In one non-limiting embodiment, a chimericNotch receptor polypeptide of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) an anti-CD19 scFv; b) a Notch receptorpolypeptide comprising: i) an LNR segment; ii) a heterodimerizationdomain (an HD-N segment and an HD-C segment); and iii) a TM domain wherethe Notch receptor polypeptide comprises one or more ligand-inducibleproteolytic cleavage sites; and c) an intracellular domain, where theintracellular domain is a Zip(−) Gal4 DNA binding polypeptide. Anexample of such a chimeric Notch receptor polypeptide is depicted inFIG. 23. In FIG. 23, the anti-CD19 scFv has the amino acid sequencedepicted in FIG. 22; the Notch receptor polypeptide includes the aminoacid sequence depicted in FIG. 16A; and the Zip(−) Gal4 DNA bindingpolypeptide has the following amino acid sequence:

(SEQ ID NO: 68) LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGKGGSGGSGGSMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQL TVSAA.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an antibody; b) a Notch receptor polypeptide comprising:i) an EGF repeat; ii) an LNR segment; iii) a heterodimerization domain(an HD-N segment and an HD-C segment); and iv) a TM domain, where theNotch receptor polypeptide comprises one or more ligand-inducibleproteolytic cleavage sites; and c) an intracellular domain, where theintracellular domain is a transcriptional activator. In one non-limitingembodiment, a chimeric Notch receptor polypeptide of the presentdisclosure comprises, in order from N-terminus to C-terminus: a) ananti-mesothelin scFv; b) a Notch receptor polypeptide comprising: i) anEGF repeat; ii) an LNR segment; iii) a heterodimerization domain (anHD-N segment and an HD-C segment); and iv) a TM domain, where the Notchreceptor polypeptide comprises one or more ligand-inducible proteolyticcleavage sites; and c) an intracellular domain, where the intracellulardomain is VP64 Zip(+) comprising an NLS. An example of such a chimericNotch receptor polypeptide is depicted in FIG. 24. In FIG. 24, theanti-mesothelin scFv has the following amino acid sequence:GSQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSSGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKS GTSPKRWIYD(SEQ ID NO:98); the Notch receptor polypeptide includes the amino acidsequence depicted in FIG. 16B; and the VP64 Zip(+) transcriptionalactivator has the following amino acid sequence:

(SEQ ID NO: 99) PKKKRKVDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSGGSGGSGGSLEIEAAFLERENTALETRVAELRQRVQRLRNR VSQYRTRYGPLGGGK.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an antibody; b) a Notch receptor polypeptide comprising:i) an LNR segment; ii) a heterodimerization domain (an HD-N segment andan HD-C segment); and iii) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa recombinase. In one non-limiting embodiment, a chimeric Notch receptorpolypeptide of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an antibody specific for a cell surfaceantigen; b) a Notch receptor polypeptide comprising: i) an LNR segment;ii) a heterodimerization domain (an HD-N segment and an HD-C segment);and iii) a TM domain, where the Notch receptor polypeptide comprises oneor more ligand-inducible proteolytic cleavage sites; and c) anintracellular domain, where the intracellular domain is a recombinase.In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an anti-CD19 scFv; b) a Notch receptor polypeptidecomprising: i) an LNR segment; ii) a heterodimerization domain (an HD-Nsegment and an HD-C segment); and iii) a TM domain where the Notchreceptor polypeptide comprises one or more ligand-inducible proteolyticcleavage sites; and c) an intracellular domain, where the intracellulardomain is a FLPe recombinase (see, e.g., Akbudak and Srivastava (2011)Mol. Biotechnol. 49:82). An example of such a chimeric Notch receptorpolypeptide is depicted in FIG. 25. In FIG. 25, the anti-CD19 scFv hasthe amino acid sequence depicted in FIG. 22; the Notch polypeptideincludes the amino acid sequence depicted in FIG. 16A; and the FLPerecombinase has the following amino acid sequence:

(SEQ ID NO: 65) MSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAAELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRYPAWNGIISQEVLDYLSSYINRRIGPVEQKLISEEDL.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an antibody; b) a Notch receptor polypeptide comprising:i) an LNR segment; ii) a heterodimerization domain (an HD-N segment andan HD-C segment); and iii) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa recombinase. In one non-limiting embodiment, a chimeric Notch receptorpolypeptide of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an antibody specific for a cell surfaceantigen; b) a Notch receptor polypeptide comprising: i) an LNR segment;ii) a heterodimerization domain (an HD-N segment and an HD-C segment);and iii) a TM domain, where the Notch receptor polypeptide comprises oneor more ligand-inducible proteolytic cleavage sites; and c) anintracellular domain, where the intracellular domain is a recombinase.In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an anti-CD19 scFv; b) a Notch receptor polypeptidecomprising: i) an LNR segment; ii) a heterodimerization domain (an HD-Nsegment and an HD-C segment); and iii) a TM domain where the Notchreceptor polypeptide comprises one or more ligand-inducible proteolyticcleavage sites; and c) an intracellular domain, where the intracellulardomain is a Cre recombinase comprising an NLS. An example of such achimeric Notch receptor polypeptide is depicted in FIG. 26. In FIG. 26,the anti-CD19 scFv has the amino acid sequence depicted in FIG. 22; theNotch polypeptide includes the amino acid sequence depicted in FIG. 16A;and the Cre recombinase has the following amino acid sequence:MVPKKKRKVSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE DGD (SEQ IDNO:100), where the Cre recombinase includes an NLS (MVPKKKRK; SEQ IDNO:84).

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an antibody; b) a Notch receptor polypeptide comprising:i) an LNR segment; ii) a heterodimerization domain (an HD-N segment andan HD-C segment); and iii) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa regulatory factor. In one non-limiting embodiment, a chimeric Notchreceptor polypeptide of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an antibody specific for a cell surfaceantigen; b) a Notch receptor polypeptide comprising: i) an LNR segment;ii) a heterodimerization domain (an HD-N segment and an HD-C segment);and iii) a TM domain, where the Notch receptor polypeptide comprises oneor more ligand-inducible proteolytic cleavage sites; and c) anintracellular domain, where the intracellular domain is a myogenicregulatory factor. In one non-limiting embodiment, a chimeric Notchreceptor polypeptide of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an anti-CD19 scFv; b) a Notch receptorpolypeptide comprising: i) an LNR segment; ii) a heterodimerizationdomain (an HD-N segment and an HD-C segment); and iii) a TM domain wherethe Notch receptor polypeptide comprises one or more ligand-inducibleproteolytic cleavage sites; and c) an intracellular domain, where theintracellular domain is a MyoD polypeptide. An example of such achimeric Notch receptor polypeptide is depicted in FIG. 27. In thisexample, MyoD is fused to a red fluorescent protein (RFP). In FIG. 27,the anti-CD19 scFv has the amino acid sequence depicted in FIG. 22; theNotch polypeptide includes the amino acid sequence depicted in FIG. 16A;and the MyoD polypeptide has the following amino acid sequence:

(SEQ ID NO: 72) MELLSPPLRDIDLTGPDGSLCSFETADDFYDDPCFDSPDLRFFEDLDPRLVHMGALLKPEEHAHFPTAVHPGPGAREDEHVRAPSGHHQAGRCLLWACKACKRKTTNADRRKAATMRERRRLSKVNEAFETLKRCTSSNPNQRLPKVEILRNAIRYIEGLQALLRDQDAAPPGAAAFYAPGPLPPGRGSEHYSGDSDASSPRSNCSDGMMDYSGPPSGPRRQNGYDTAYYSEAARESRPGKSAAVSSLDCLSSIVERISTDSPAAPALLLADAPPESPPGPPEGASLSDTEQGTQTPSPD AAPQCPAGSNPNAIYQVL.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises, in order from N-terminus toC-terminus: a) an antibody; b) a Notch receptor polypeptide comprising:i) an LNR segment; ii) a heterodimerization domain (an HD-N segment andan HD-C segment); and iii) a TM domain, where the Notch receptorpolypeptide comprises one or more ligand-inducible proteolytic cleavagesites; and c) an intracellular domain, where the intracellular domain isa transcription factor. In one non-limiting embodiment, a chimeric Notchreceptor polypeptide of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an antibody specific for a cell surfaceantigen; b) a Notch receptor polypeptide comprising: i) an LNR segment;ii) a heterodimerization domain (an HD-N segment and an HD-C segment);and iii) a TM domain, where the Notch receptor polypeptide comprises oneor more ligand-inducible proteolytic cleavage sites; and c) anintracellular domain, where the intracellular domain is aT-box-containing transcription factor. In one non-limiting embodiment, achimeric Notch receptor polypeptide of the present disclosure comprises,in order from N-terminus to C-terminus: a) an anti-CD19 scFv; b) a Notchreceptor polypeptide comprising: i) an LNR segment; ii) aheterodimerization domain (an HD-N segment and an HD-C segment); andiii) a TM domain where the Notch receptor polypeptide comprises one ormore ligand-inducible proteolytic cleavage sites; and c) anintracellular domain, where the intracellular domain is a Tbx21polypeptide (also known as Tbet (GenBank BC039739)). An example of sucha chimeric Notch receptor polypeptide is depicted in FIG. 28. Tbx21protein is a Th1 cell-specific transcription factor that controls theexpression of interferon-gamma, a Th1 cytokine. In FIG. 28, theanti-CD19 scFv has the amino acid sequence depicted in FIG. 22; theNotch polypeptide includes the amino acid sequence depicted in FIG. 16A;and the Tbx21 protein has the following amino acid sequence:

(SEQ ID NO: 71) MGIVEPGCGDMLTGTEPMPGSDEGRAPGADPQHRYFYPEPGAQDADERRGGGSLGSPYPGGALVPAPPSRFLGAYAYPPRPQAAGFPGAGESFPPPADAEGYQPGEGYAAPDPRAGLYPGPREDYALPAGLEVSGKLRVALNNHLLWSKFNQHQTEMIITKQGRRMFPFLSFTVAGLEPTSHYRMFVDVVLVDQHHWRYQSGKWVQCGKAEGSMPGNRLYVHPDSPNTGAHWMRQEVSFGKLKLTNNKGASNNVTQMIVLQSLHKYQPRLHIVEVNDGEPEAACNASNTHIFTFQETQFIAVTAYQNAEITQLKIDNNPFAKGFRENFESMYTSVDTSIPSPPGPNCQFLGGDHYSPLLPNQYPVPSRFYPDLPGQAKDVVPQAYWLGAPRDHSYEAEFRAVSMKPAFLPSAPGPTMSYYRGQEVLAPGAGWPVAPQYPPKMGPASWFRPMRTLPMEPGPGGSEGRGPEDQGPPLVWTEIAPIRPESSDSGLGEGDSKRRRVSPYPSSGDSSSPAGAPSPFDKEAEGQFYNYFPN.

In one non-limiting embodiment, a chimeric Notch receptor polypeptide ofthe present disclosure comprises: a) an extracellular domain; b) a Notchreceptor polypeptide that comprises the following amino acid sequence:IPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRRQLCIQKL (SEQ ID NO:4);where the TM domain is underlined; where the Notch receptor polypeptidecomprises an S2 proteolytic cleavage site and an S3 proteolytic cleavagesite; and c) an intracellular domain. In one non-limiting embodiment, achimeric Notch receptor polypeptide of the present disclosure comprises:a) an extracellular domain; b) a Notch receptor polypeptide thatcomprises the following amino acid sequence:IPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRRQLCIQKL (SEQ ID NO:4);where the TM domain is underlined; where the Notch receptor polypeptidecomprises an S2 proteolytic cleavage site and an S3 proteolytic cleavagesite; and c) an intracellular domain, where the intracellular domain isa transcriptional activator. In one non-limiting embodiment, a chimericNotch receptor polypeptide of the present disclosure comprises: a) anextracellular domain, where the extracellular domain is a polypeptidefound on the surface of immune cells (T cells, monocytes, macrophages,and dendritic cells); b) a Notch receptor polypeptide that comprises thefollowing amino acid sequence:IPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRRQLCIQKL (SEQ ID NO:4);where the TM domain is underlined; where the Notch receptor polypeptidecomprises an S2 proteolytic cleavage site and an S3 proteolytic cleavagesite; and c) an intracellular domain, where the intracellular domain isa transcriptional activator. In one non-limiting embodiment, a chimericNotch receptor polypeptide of the present disclosure comprises: a) anextracellular domain, where the extracellular domain is a CD4extracellular domain; b) a Notch receptor polypeptide that comprises thefollowing amino acid sequence:IPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRRQLCIQKL (SEQ ID NO:4);where the TM domain is underlined; where the Notch receptor polypeptidecomprises an S2 proteolytic cleavage site and an S3 proteolytic cleavagesite; and c) an intracellular domain, where the intracellular domain isa tTA transcriptional activator. An example of such a chimeric Notchreceptor polypeptide is depicted in FIG. 29.

Nucleic Acids

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a chimeric Notch receptor polypeptide of the presentdisclosure. In some cases, a nucleic acid comprising a nucleotidesequence encoding a chimeric Notch receptor polypeptide of the presentdisclosure is contained within an expression vector. Thus, the presentdisclosure provides a recombinant expression vector comprising a nucleicacid comprising a nucleotide sequence encoding a chimeric Notch receptorpolypeptide of the present disclosure. In some cases, the nucleotidesequence encoding a chimeric Notch receptor polypeptide of the presentdisclosure is operably linked to a transcriptional control element(e.g., a promoter; an enhancer; etc.). In some cases, thetranscriptional control element is inducible. In some cases, thetranscriptional control element is constitutive. In some cases, thepromoters are functional in eukaryotic cells. In some cases, thepromoters are cell type-specific promoters. In some cases, the promotersare tissue-specific promoters.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation control elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc. may be used in the expression vector(see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

A promoter can be a constitutively active promoter (i.e., a promoterthat is constitutively in an active/“ON” state), it may be an induciblepromoter (i.e., a promoter whose state, active/“ON” or inactive/“OFF”,is controlled by an external stimulus, e.g., the presence of aparticular temperature, compound, or protein.), it may be a spatiallyrestricted promoter (i.e., transcriptional control element, enhancer,etc.)(e.g., tissue specific promoter, cell type specific promoter,etc.), and it may be a temporally restricted promoter (i.e., thepromoter is in the “ON” state or “OFF” state during specific stages ofembryonic development or during specific stages of a biological process,e.g., hair follicle cycle in mice).

Suitable promoter and enhancer elements are known in the art. Forexpression in a bacterial cell, suitable promoters include, but are notlimited to, lacd, lacZ, T3, T7, gpt, lambda P and trc. For expression ina eukaryotic cell, suitable promoters include, but are not limited to,light and/or heavy chain immunoglobulin gene promoter and enhancerelements; cytomegalovirus immediate early promoter; herpes simplex virusthymidine kinase promoter; early and late SV40 promoters; promoterpresent in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters.

Suitable reversible promoters, including reversible inducible promotersare known in the art. Such reversible promoters may be isolated andderived from many organisms, e.g., eukaryotes and prokaryotes.Modification of reversible promoters derived from a first organism foruse in a second organism, e.g., a first prokaryote and a second aeukaryote, a first eukaryote and a second a prokaryote, etc., is wellknown in the art. Such reversible promoters, and systems based on suchreversible promoters but also comprising additional control proteins,include, but are not limited to, alcohol regulated promoters (e.g.,alcohol dehydrogenase I (alcA) gene promoter, promoters responsive toalcohol transactivator proteins (AlcR), etc.), tetracycline regulatedpromoters, (e.g., promoter systems including TetActivators, TetON,TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoidreceptor promoter systems, human estrogen receptor promoter systems,retinoid promoter systems, thyroid promoter systems, ecdysone promotersystems, mifepristone promoter systems, etc.), metal regulated promoters(e.g., metallothionein promoter systems, etc.), pathogenesis-relatedregulated promoters (e.g., salicylic acid regulated promoters, ethyleneregulated promoters, benzothiadiazole regulated promoters, etc.),temperature regulated promoters (e.g., heat shock inducible promoters(e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), lightregulated promoters, synthetic inducible promoters, and the like.

Inducible promoters suitable for use include any inducible promoterdescribed herein or known to one of ordinary skill in the art. Examplesof inducible promoters include, without limitation,chemically/biochemically-regulated and physically-regulated promoterssuch as alcohol-regulated promoters, tetracycline-regulated promoters(e.g., anhydrotetracycline (aTc)-responsive promoters and othertetracycline-responsive promoter systems, which include a tetracyclinerepressor protein (tetR), a tetracycline operator sequence (tetO) and atetracycline transactivator fusion protein (tTA)), steroid-regulatedpromoters (e.g., promoters based on the rat glucocorticoid receptor,human estrogen receptor, moth ecdysone receptors, and promoters from thesteroid/retinoid/thyroid receptor superfamily), metal-regulatedpromoters (e.g., promoters derived from metallothionein (proteins thatbind and sequester metal ions) genes from yeast, mouse and human),pathogenesis-regulated promoters (e.g., induced by salicylic acid,ethylene or benzothiadiazole (BTH)), temperature/heat-induciblepromoters (e.g., heat shock promoters), and light-regulated promoters(e.g., light responsive promoters from plant cells).

In some cases, the promoter is a CD8 cell-specific promoter, a CD4cell-specific promoter, a neutrophil-specific promoter, or anNK-specific promoter. For example, a CD4 gene promoter can be used; see,e.g., Salmon et al. (1993) Proc. Nat. Acad. Sci. USA 90: 7739; andMarodon et al. (2003) Blood 101:3416. As another example, a CD8 genepromoter can be used. NK cell-specific expression can be achieved by useof an Ncr1 (p46) promoter; see, e.g., Eckelhart et al. (2011) Blood117:1565.

In some cases, the promoter is a cardiomyocyte-specific promoter. Insome cases, the promoter is a smooth muscle cell-specific promoter. Insome cases, the promoter is a neuron-specific promoter. In some cases,the promoter is an adipocyte-specific promoter. Other cell type-specificpromoters are known in the art and are suitable for use herein.

In some cases, a nucleic acid comprising a nucleotide sequence encodinga chimeric Notch receptor polypeptide of the present disclosure is arecombinant expression vector. In some embodiments, the recombinantexpression vector is a viral construct, e.g., a recombinantadeno-associated virus (AAV) construct, a recombinant adenoviralconstruct, a recombinant lentiviral construct, a recombinant retroviralconstruct, etc. In some cases, a nucleic acid comprising a nucleotidesequence encoding a chimeric Notch receptor polypeptide of the presentdisclosure is a recombinant lentivirus vector. In some cases, a nucleicacid comprising a nucleotide sequence encoding a chimeric Notch receptorpolypeptide of the present disclosure is a recombinant AAV vector.

Suitable expression vectors include, but are not limited to, viralvectors (e.g. viral vectors based on vaccinia virus; poliovirus;adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549,1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS92:7700 7704, 1995; Sakamoto et al., Hum Gene Ther 5:1088 1097, 1999; WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali etal., Hum Mol Genet 5:591594, 1996; Srivastava in WO 93/09239, Samulskiet al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40;herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshiet al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816,1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosisvirus, and vectors derived from retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus); and the like. In some cases, the vector is a lentivirusvector. Also suitable are transposon-mediated vectors, such as piggybackand sleeping beauty vectors.

Host Cells

The present disclosure provides host cells genetically modified with anucleic acid of the present disclosure, i.e., host cells geneticallymodified with a nucleic acid comprising a nucleotide sequence encoding achimeric Notch receptor polypeptide of the present disclosure. Thepresent disclosure provides a method of modulating an activity of a cellthat expresses a chimeric Notch polypeptide of the present disclosure.The method generally involves contacting the cell with a second memberof the specific binding pair. Binding of the first member of thespecific binding pair to the second member of the specific binding pairinduces cleavage of the Notch receptor polypeptide at the one or moreligand-inducible proteolytic cleavage sites, thereby releasing theintracellular domain. Release of the intracellular domain modulates anactivity of the cell.

In some cases, the cell is a eukaryotic cell. In some cases, the cell isa mammalian cell, an amphibian cell, a reptile cell, an avian cell, or aplant cell. In some cases, the cell is a plant cell.

In some cases, the cell is a mammalian cell. In some cases, the cell isa human cell. In some cases, the cell is a mouse cell. In some cases,the cell is rat cell. In some cases, the cell is non-human primate cell.In some cases, the cell is lagomorph cell. In some cases, the cell is anungulate cell.

In some cases, the cell is an immune cell, e.g., a T cell, a B cell, amacrophage, a dendritic cell, a natural killer cell, a monocyte, etc. Insome cases, the cell is a T cell. In some cases, the cell is a cytotoxicT cell (e.g., a CD8⁺ T cell). In some cases, the cell is a helper T cell(e.g., a CD4⁺ T cell). In some cases, the cell is a regulatory T cell(“Treg”). In some cases, the cell is a B cell. In some cases, the cellis a macrophage. In some cases, the cell is a dendritic cell. In somecases, the cell is a peripheral blood mononuclear cell. In some cases,the cell is a monocyte. In some cases, the cell is a natural killer (NK)cell. In some cases, the cell is a CD4⁺, FOXP3⁺ Treg cell. In somecases, the cell is a CD4⁺, FOXP3⁻ Treg cell.

In some instances, the cell is obtained from an individual. For example,in some cases, the cell is a primary cell. As another example, the cellis a stem cell or progenitor cell obtained from an individual.

As one non-limiting example, in some cases, the cell is an immune cellobtained from an individual. As an example, the cell can be a Tlymphocyte obtained from an individual. As another example, the cell isa cytotoxic cell (e.g., a cytotoxic T cell) obtained from an individual.As another example, the cell can be a helper T cell obtained from anindividual. As another example, the cell can be a regulatory T cellobtained from an individual. As another example, the cell can be an NKcell obtained from an individual. As another example, the cell can be amacrophage obtained from an individual. As another example, the cell canbe a dendritic cell obtained from an individual. As another example, thecell can be a B cell obtained from an individual. As another example,the cell can be a peripheral blood mononuclear cell obtained from anindividual.

In some cases, the host cell is a somatic cell, e.g. a fibroblast, ahematopoietic cell, a neuron, a pancreatic cell, a muscle cell, a bonecell, a hepatocyte, a pancreatic cell, an epithelial cell, anendothelial cell, a cardiomyocyte, a T cell, a B cell, an osteocyte, andthe like.

In some cases, the cell is genetically modified to express two differentchimeric Notch receptor polypeptides of the present disclosure. Forexample, in some cases, a host cell is genetically modified to express:i) a first chimeric Notch receptor polypeptide comprising a first memberof a first specific binding pair; and ii) at least a second chimericNotch receptor polypeptide comprising a first member of a secondspecific binding pair, where the first and the second specific bindingpairs are different from one another such that binding of a secondmember of the first specific binding pair to the first member of thefirst specific binding pair does not result in release of theintracellular domain of the second chimeric Notch receptor polypeptide,and such that binding of a second member of the second specific bindingpair to the first member of the second specific binding pair does notresult in release of the intracellular domain of the first secondchimeric Notch receptor polypeptide.

In some cases, the cell is genetically modified to express a chimericNotch receptor polypeptide of the present disclosure. In some cases, thecell is genetically modified to express a chimeric Notch receptorpolypeptide of the present disclosure; and is further geneticallymodified to express a chimeric antigen receptor (CAR). For example, insome cases, the host cell is genetically modified with a nucleic acidcomprising a nucleotide sequence encoding a CAR, and the intracellulardomain of the chimeric polypeptide is a transcriptional activator. Insome cases, the nucleotide sequence encoding the CAR is operably linkedto a transcriptional control element that is activated by theintracellular domain of the chimeric polypeptide. Many CAR polypeptideshave been described in the art, any of which is suitable for use herein.

In some cases, the CAR comprises an extracellular domain, atransmembrane region and an intracellular signaling domain; where theextracellular domain comprises a ligand or a receptor linked to anoptional support region capable of tethering the extracellular domain toa cell surface, and the intracellular signaling domain comprises thesignaling domain from the zeta chain of the human CD3 complex (CD3zeta)and one or more costimulatory signaling domains, such as those fromCD28, 4-1BB and OX-40. The extracellular domain contains a recognitionelement (e.g., an antibody or other target-binding scaffold) thatenables the CAR to bind a target. In some cases, a CAR comprises theantigen binding domains of an antibody (e.g., an scFv) linked to T-cellsignaling domains. In some cases, when expressed on the surface of a Tcell, the CAR can direct T cell activity to those cells expressing areceptor or ligand for which this recognition element is specific. As anexample, a CAR that contains an extracellular domain that contains arecognition element specific for a tumor antigen can direct T cellactivity to tumor cells that bear the tumor antigen. The intracellularregion enables the cell (e.g., a T cell) to receive costimulatorysignals. The costimulatory signaling domains can be selected from CD28,4-1BB, OX-40 or any combination of these. Exemplary CARs comprise ahuman CD4 transmembrane region, a human IgG4 Fc and a receptor or ligandthat is tumor-specific, such as an IL13 or IL3 molecule.

The extracellular domain is made up of a soluble receptor ligand (thatis specific for a target tumor antigen or other tumor cell-surfacemolecule) linked to an optional support region capable of tethering theextracellular domain to a cell surface In some cases, the CAR is aheterodimeric, conditionally active CAR, as described in WO 2014/127261.In some embodiments, the heterodimeric, conditionally active CAR isactivated by: i) binding an antigen for which the CAR is specific; andii) a dimerizing agent that induces dimerization of the two polypeptidechains of the heterodimeric, conditionally active CAR. The dimerizingagent can be a small molecule, or can be light.

Transgenic Organisms

The present disclosure provides non-human transgenic organisms thatcomprise a nucleic acid encoding a chimeric Notch polypeptide of thepresent disclosure. A transgenic non-human organism of the presentdisclosure comprises a genome that has been genetically modified toinclude a nucleic acid comprising a nucleotide sequence encoding achimeric Notch polypeptide of the present disclosure.

Methods of producing genetically modified organisms are known in theart. For example, see Cho et al., Curr Protoc Cell Biol. 2009 March;Chapter 19:Unit 19.11: Generation of transgenic mice; Gama et al., BrainStruct Funct. 2010 March; 214(2-3):91-109. Epub 2009 Nov. 25: Animaltransgenesis: an overview; and Husaini et al., GM Crops. 2011June-December; 2(3):150-62. Epub 2011 Jun. 1: Approaches for genetargeting and targeted gene expression in plants.

In a non-human transgenic organism of the present disclosure, a nucleicacid comprising a nucleotide sequence encoding a chimeric Notchpolypeptide of the present disclosure can be under the control of (i.e.,operably linked to) an unknown promoter (e.g., when the nucleic acidrandomly integrates into a host cell genome) or can be under the controlof (i.e., operably linked to) a known promoter. Suitable known promoterscan be any known promoter and include constitutively active promoters(e.g., CMV promoter), inducible promoters (e.g., heat shock promoter,Tetracycline-regulated promoter, Steroid-regulated promoter,Metal-regulated promoter, estrogen receptor-regulated promoter, etc.),spatially restricted and/or temporally restricted promoters (e.g., atissue specific promoter, a cell type specific promoter, etc.), etc.

A subject genetically modified organism (e.g. an organism whose genomecomprises a nucleotide sequence encoding chimeric Notch polypeptide ofthe present disclosure can be any organism including for example, aplant; an invertebrate (e.g., a cnidarian, an echinoderm, a worm, a fly,etc.); a non-mammalian vertebrate (e.g., a fish (e.g., zebrafish, pufferfish, gold fish, etc.)); an amphibian (e.g., salamander, frog, etc.); areptile; a bird; a mammal; etc.); an ungulate (e.g., a goat, a pig, asheep, a cow, etc.); a rodent (e.g., a mouse, a rat, a hamster, a guineapig); a lagomorph (e.g., a rabbit); etc. In some cases, the transgenicnon-human organism is a mouse. In some cases, the transgenic non-humanorganism is a rat. In some cases, the transgenic non-human organism is aplant.

In some embodiments, the transgenic non-human animal is homozygous forthe transgene encoding a chimeric Notch polypeptide of the presentdisclosure. In some embodiments, the transgenic non-human animal isheterozygous for the transgene encoding a chimeric Notch polypeptide ofthe present disclosure.

Methods

A chimeric Notch receptor polypeptide of the present disclosure, and anucleic acid of the present disclosure (a nucleic acid comprising anucleotide sequence encoding a chimeric Notch receptor polypeptide), anda recombinant expression vector comprising a nucleic acid of the presentdisclosure, are useful in a variety of applications. The presentdisclosure provides such applications.

The present disclosure provides a method of modulating an activity of acell that expresses a chimeric Notch polypeptide of the presentdisclosure. Methods of the present disclosure for modulating theactivity of a cell can be carried out in vitro, ex vivo, or in vivo.Methods of the present disclosure for modulating the activity of a cellcan be carried out in a single cell, or in a multicellular environment(e.g., a naturally-occurring tissue; an artificial tissue; etc.).Methods of the present disclosure for modulating the activity of a cellcan be carried out in parallel or in series.

Methods of Modulating an Activity of a Cell

The present disclosure provides a method of modulating an activity of acell that expresses a chimeric Notch polypeptide of the presentdisclosure. In some cases, the method comprises: contacting the cellwith a second member of the specific binding pair, wherein binding ofthe first member of the specific binding pair to the second member ofthe specific binding pair induces cleavage of the Notch receptorpolypeptide at the one or more ligand-inducible proteolytic cleavagesites, thereby releasing the intracellular domain, wherein release ofthe intracellular domain modulates the activity of the cell. Theintracellular domain provides an “effector function,” where an effectorfunction can be transcriptional activation; transcriptional repression;translational activation; translational repression; modulation oforganelle function; immune cell activation; immune cell repression;induction of apoptosis; repression of apoptosis; nuclease activity;regulation of differentiation; replacement of a target nucleic acid;modification of a target nucleic acid; etc. Activities of a cell thatcan be modulated using a method of the present disclosure include, butare not limited to, immune cell activation (e.g., T cell activation,etc.); apoptosis; production of effector molecules (e.g., cytokines,antibodies, growth factors, etc.); transcription of a target nucleicacid; translation of a target mRNA; organelle activity; intracellulartrafficking; differentiation; and the like. The methods of the presentdisclosure can also be used to cause the release of effectors that actat the plasma membrane, thereby leading to modification of cellularactivity (e.g. release of immune co-inhibitory receptor motifs thatprovide for immune activation).

The present disclosure provides a method of inducing an effectorfunction in a cell that expresses a chimeric Notch polypeptide of thepresent disclosure. In some cases, the method comprises: contacting thecell with a second member of the specific binding pair, wherein bindingof the first member of the specific binding pair to the second member ofthe specific binding pair induces cleavage of the Notch receptorpolypeptide at the one or more ligand-inducible proteolytic cleavagesites, thereby releasing the intracellular domain, wherein theintracellular domain provides an effector function, wherein release ofthe intracellular domain provides for action of the effector function inthe cell.

In some cases, binding of the first member of the specific binding pairto the second member of the specific binding pair induces cleavage ofthe Notch receptor polypeptide at the one or more ligand-inducibleproteolytic cleavage sites, thereby releasing the intracellular domain,wherein the intracellular domain is an apoptosis inducer. In some cases,binding of the first member of the specific binding pair to the secondmember of the specific binding pair induces cleavage of the Notchreceptor polypeptide at the one or more ligand-inducible proteolyticcleavage sites, thereby releasing the intracellular domain, wherein theintracellular domain is a recombinase. In some cases, binding of thefirst member of the specific binding pair to the second member of thespecific binding pair induces cleavage of the Notch receptor polypeptideat the one or more ligand-inducible proteolytic cleavage sites, therebyreleasing the intracellular domain, wherein the intracellular domain isa Cas9 polypeptide. In some cases, binding of the first member of thespecific binding pair to the second member of the specific binding pairinduces cleavage of the Notch receptor polypeptide at the one or moreligand-inducible proteolytic cleavage sites, thereby releasing theintracellular domain, wherein the intracellular domain is a dCas9polypeptide. In some cases, binding of the first member of the specificbinding pair to the second member of the specific binding pair inducescleavage of the Notch receptor polypeptide at the one or moreligand-inducible proteolytic cleavage sites, thereby releasing theintracellular domain, wherein the intracellular domain is atranscriptional activator. In some cases, binding of the first member ofthe specific binding pair to the second member of the specific bindingpair induces cleavage of the Notch receptor polypeptide at the one ormore ligand-inducible proteolytic cleavage sites, thereby releasing theintracellular domain, wherein the intracellular domain is atranscription repressor.

The methods of the present disclosure can be carried out in vivo, invitro, or ex vivo.

In some cases, a method of the present disclosure is carried out exvivo, where cells are obtained from an individual, and geneticallymodified to express: i) a single chimeric Notch receptor polypeptide ofthe present disclosure; ii) two or more chimeric Notch receptorpolypeptides of the present disclosure; iii) a chimeric Notch receptorpolypeptide of the present disclosure and a CAR; or iv) a chimeric Notchreceptor polypeptide of the present disclosure and a naturally-occurringor synthetic receptor that provides for a non-endogenous responsivecapability in the cell.

As one non-limiting example, the present disclosure provides a method oftreating cancer in an individual having a cancer, the method comprising:i) genetically modifying T lymphocytes or natural killer (NK) cellsobtained from the individual with an expression vector comprising anucleotide sequence encoding a chimeric Notch receptor polypeptide ofthe present disclosure, where the chimeric Notch receptor polypeptide isspecific for an epitope on a cancer cell in the individual, and wherethe genetic modification is carried out ex vivo; ii) introducing thegenetically modified T lymphocytes or NK cells into the individual,where the genetically modified T lymphocytes or NK recognize and killthe cancer cell, thereby treating the cancer.

In some cases, a method of the present disclosure is carried out invivo, e.g., where an expression vector comprising a nucleotide sequenceencoding a chimeric Notch receptor polypeptide of the present disclosureis administered to an individual in need thereof. Methods ofadministering an expression vector to an individual are well known inthe art; any known method for administering an expression vector to anindividual is suitable for use in a method of the present disclosure.

In some cases, a method of the present disclosure is carried out invitro, e.g., in in vitro cell culture, with cells grown as single cellsin suspension, with cells grown on a solid support, with cells grown ina 3-dimensional scaffold, and the like.

Direct Control of Effector Function

The present disclosure provides a method of modulating an activity of acell that expresses a chimeric Notch polypeptide of the presentdisclosure. As described in detail above, the chimeric Notch polypeptidecomprises an extracellular domain that comprises a first member of aspecific binding pair. The chimeric Notch polypeptide also comprises aNotch receptor polypeptide comprising one or more ligand-inducibleproteolytic cleavage sites; and an intracellular domain. In some cases,the method comprises contacting the cell with a second member of thespecific binding pair, where binding of the first member of the specificbinding pair to the second member of the specific binding pair inducescleavage of the Notch receptor polypeptide at the one or moreligand-inducible proteolytic cleavage sites, thereby releasing theintracellular domain, wherein release of the intracellular domainmodulates the activity of the cell. In some cases, the second member ofthe specific binding pair is present on the cell surface of a secondcell that contacts the cell that expresses a chimeric Notch polypeptideof the present disclosure. In some cases, the second member of thespecific binding pair is soluble.

In some cases, the released intracellular domain directly modulates thecell that expresses a chimeric Notch polypeptide of the presentdisclosure. Such an embodiment is illustrated schematically in FIG. 5.

As a non-limiting example of a direct control of effector function, achimeric Notch receptor polypeptide of the present disclosure comprisesan extracellular domain comprising a scFv specific for a ligand, and anapoptotic regulator (e.g., tBID) as the intracellular domain. Thechimeric Notch receptor polypeptide is expressed on the surface of afirst cell. Upon binding to a second member of the specific binding pair(which in this case is a ligand (antigen) specifically bound by thescFv) on the surface of a second cell, the tBID is released in the firstcell and induces apoptosis in the first cell. This example isillustrated schematically in FIG. 6.

Indirect Control of Effector Function

The present disclosure provides a method of modulating an activity of acell that expresses a chimeric Notch polypeptide of the presentdisclosure. As described in detail above, the chimeric Notch polypeptidecomprises an extracellular domain that comprises a first member of aspecific binding pair. The chimeric Notch polypeptide also comprises aNotch receptor polypeptide comprising one or more ligand-inducibleproteolytic cleavage sites; and an intracellular domain. In some cases,the method comprises contacting the cell with a second member of thespecific binding pair, where binding of the first member of the specificbinding pair to the second member of the specific binding pair inducescleavage of the Notch receptor polypeptide at the one or moreligand-inducible proteolytic cleavage sites, thereby releasing theintracellular domain. In some cases, the second member of the specificbinding pair is present on the cell surface of a second cell thatcontacts the cell that expresses a chimeric Notch polypeptide of thepresent disclosure. In some cases, the second member of the specificbinding pair is soluble.

In some cases, the released intracellular domain is a transcriptionfactor or a translation factor that, when released, regulates expression(e.g., increases transcription; decreases transcription; increasetranslation; decreases translation; etc.) of a target nucleic acid. Insome cases, the released intracellular domain is a transcription factorthat, when released, induces transcription of an effector gene,resulting in production of a gene product (e.g., an effectorpolypeptide; an effector nucleic acid) encoded by the effector gene. Anexample of such an “indirect” control of effector function is depictedin FIG. 7. In some cases, the effector polypeptide is an apoptosisinducer, an activating immunoreceptor, an inhibiting immunoreceptor, atranscription factor, an apoptosis inhibitor, a secreted factor (e.g., acytokine; a hormone; a chemokine; an antibody; a receptor that altersthe ability of the cell to respond to one or more endogenous factors; adominant negative regulatory protein; an intracellular blocking protein;etc.) or a site-specific nuclease. In some cases, the releasedintracellular domain is a polypeptide that, when released, modulates(increases or decreases) translation, mRNA stability, or proteinprocessing, of a target gene product, where the target gene productprovides an effector function.

As a non-limiting example of indirect control of effector function, achimeric Notch receptor polypeptide of the present disclosure comprisesan extracellular domain comprising a scFv specific for a ligand, and anintracellular domain comprising a transcription factor (e.g., tTa). Thechimeric Notch receptor polypeptide is expressed on the surface of afirst cell. Upon binding to a second member of the specific binding pair(which in this case is a ligand (antigen) specifically bound by thescFv) on the surface of a second cell, the transcription factor (in thiscase, tTa) is released in the first cell and induces transcription of anucleic acid encoding an apoptosis inducer (in this case, tBID). ThetBID is produced in the first cell, and induces apoptosis in the firstcell. This example is illustrated schematically in FIG. 8.

Use of Chimeric Notch Receptor Polypeptides in the Targeted Delivery andSecretion of Biologic Agents

A chimeric Notch receptor polypeptide of the present disclosure can beused to control expression and secretion of biological molecules, suchas cytokines, growth factors, antibodies, and other binding, agonist,trapping, or blocking agents that are genetically encoded.

Binding of a chimeric Notch receptor (or combinations of chimeric Notchreceptors that work cooperatively) can be used to sense a particularregion, tissue or cell type in the body, which then triggers thelocalized expression/delivery of the secreted biologic to that site.Control of delivery of the biologic could be via indirect control(control of transcription of the agent), or via control of otherprocesses involved in expression, processing and secretion of thebiologic.

Combinatorial Use of Chimeric Notch Receptor Polypeptides—MultipleReceptors in Parallel; a Chimeric Notch Polypeptide and a CAR

The present disclosure provides a method of modulating an activity of acell that expresses: i) a chimeric Notch polypeptide of the presentdisclosure; and b) a chimeric antigen receptor. The method involvescontacting a cell, which expresses both a chimeric Notch receptorpolypeptide of the present disclosure and a CAR, with: i) a secondmember of the specific binding pair (where the second member of thespecific binding pair binds to the first member, present in the chimericNotch receptor polypeptide, of the specific binding pair); and ii) theantigen to which the CAR binds. In these embodiments, modulation ofactivity of the cell requires both a second member of the specificbinding pair, and the antigen to which the CAR specifically binds.

In some cases, the CAR comprises an extracellular domain, atransmembrane region and an intracellular signaling domain; where theextracellular domain comprises a ligand or a receptor linked to anoptional support region capable of tethering the extracellular domain toa cell surface, and the intracellular signaling domain comprises thesignaling domain from the zeta chain of the human CD3 complex (CD3zeta)and one or more costimulatory signaling domains, such as those fromCD28, 4-1BB and OX-40. The extracellular domain contains a recognitionelement (e.g., an antibody or other target-binding scaffold) thatenables the CAR to bind a target. In some cases, a CAR comprises theantigen binding domains of an antibody (e.g., an scFv) linked to T-cellsignaling domains. In some cases, when expressed on the surface of a Tcell, the CAR can direct T cell activity to those cells expressing areceptor or ligand for which this recognition element is specific. As anexample, a CAR that contains an extracellular domain that contains arecognition element specific for a tumor antigen can direct T cellactivity to tumor cells that bear the tumor antigen. The intracellularregion enables the cell (e.g., a T cell) to receive costimulatorysignals. The costimulatory signaling domains can be selected from CD28,4-1BB, OX-40 or any combination of these. Exemplary CARs comprise ahuman CD4 transmembrane region, a human IgG4 Fc and a receptor or ligandthat is tumor-specific, such as an IL13 or IL3 molecule.

The extracellular domain is made up of a soluble receptor ligand (thatis specific for a target tumor antigen or other tumor cell-surfacemolecule) linked to an optional support region capable of tethering theextracellular domain to a cell surface In some cases, the CAR is aheterodimeric, conditionally active CAR, as described in WO 2014/127261.

The chimeric notch receptor can also be used to similarly modulate theactivity of any other natural, chimeric, or orthogonal receptor whoseactivity is not constitutively present in the cell, or whose activity isnot normally present in the cell, thereby altering the signals the cellresponds to.

Combinatorial Use of Chimeric Notch Receptor Polypeptides—MultipleReceptors in Parallel; Two Different Chimeric Notch Polypeptides

The present disclosure provides a method of modulating an activity of acell that expresses: i) a first chimeric Notch polypeptide of thepresent disclosure; and b) a second chimeric Notch polypeptide of thepresent disclosure. For example, in some cases, the cell expresses: i) afirst chimeric Notch receptor polypeptide comprising a first member of afirst specific binding pair; and ii) at least a second chimeric Notchreceptor polypeptide comprising a first member of a second specificbinding pair, where the first and the second specific binding pairs aredifferent from one another such that binding of a second member of thefirst specific binding pair to the first member of the first specificbinding pair does not result in release of the intracellular domain ofthe second chimeric Notch receptor polypeptide, and such that binding ofa second member of the second specific binding pair to the first memberof the second specific binding pair does not result in release of theintracellular domain of the first second chimeric Notch receptorpolypeptide. In these embodiments, the intracellular domain of the firstchimeric Notch receptor polypeptide provides a first effector function;and the intracellular domain of the second chimeric Notch receptorpolypeptide provides a second effector function that is different fromthe first effector function. A schematic illustration of theseembodiments is presented in FIG. 9A.

The present disclosure provides a method of modulating an activity of acell that expresses: i) a first chimeric Notch polypeptide of thepresent disclosure; and b) a second chimeric Notch polypeptide of thepresent disclosure. For example, in some cases, the cell expresses: i) afirst chimeric Notch receptor polypeptide comprising a first member of afirst specific binding pair; and ii) at least a second chimeric Notchreceptor polypeptide comprising a first member of a second specificbinding pair, where the first and the second specific binding pairs aredifferent from one another such that binding of a second member of thefirst specific binding pair to the first member of the first specificbinding pair does not result in release of the intracellular domain ofthe second chimeric Notch receptor polypeptide, and such that binding ofa second member of the second specific binding pair to the first memberof the second specific binding pair does not result in release of theintracellular domain of the first second chimeric Notch receptorpolypeptide. In these embodiments, the released intracellular domain ofthe first chimeric Notch receptor polypeptide binds to (or otherwiseoperably interacts with) the released intracellular domain of the secondchimeric Notch receptor polypeptide to provide an effector function. Thereleased intracellular domain of the first chimeric Notch receptorpolypeptide by itself does not provide the effector function; and thereleased intracellular domain of the second chimeric Notch receptorpolypeptide by itself does not provide the effector function. However,the two released intracellular domains together provide an effectorfunction. A schematic illustration of these embodiments is presented inFIG. 9A.

A similar embodiment could utilize indirect regulation by the twodifferent chimeric Notch receptors, whereby each of the two differentchimeric Notch receptors would induce the expression of effectors that,only when expressed together, would induce the effector function.

A chimeric Notch receptor of the present disclosure can also be used tosimilarly modulate the activity of any other natural, chimeric, ororthogonal receptor whose activity is not constitutively present in thecell or not normally present in the cell.

Combinatorial Use of Chimeric Notch Receptor Polypeptides—MultipleReceptors in Series; a Chimeric Notch Polypeptide and a CAR

The present disclosure provides a method of modulating an activity of acell that is genetically modified with: a) a nucleic acid comprising anucleotide sequence encoding a chimeric Notch polypeptide of the presentdisclosure; and b) a nucleic acid comprising a nucleotide sequenceencoding a chimeric antigen receptor, where the nucleotide sequenceencoding the chimeric antigen receptor is under control of an induciblepromoter. The method involves: i) contacting the cell, which expressesthe chimeric Notch receptor polypeptide of the present disclosure (butwhich does not express the CAR), with a second member of the specificbinding pair (where the second member of the specific binding pair bindsto the first member, present in the chimeric Notch receptor polypeptide,of the specific binding pair), where contacting the cell with the secondmember of the specific binding pair induces release of the intracellulardomain of the chimeric Notch receptor polypeptide, where theintracellular domain is a transcription factor that activatestranscription of the nucleic acid comprising a nucleotide sequenceencoding a chimeric antigen receptor, resulting in expression of theCAR; and ii) after the contacting step of (i), contacting the cell withthe antigen to which the CAR binds. The second contacting step resultsin modulation of activity by the CAR. In these embodiments, modulationof activity of the cell requires both a second member of the specificbinding pair, and the antigen to which the CAR specifically binds. Anexample of these embodiments is illustrated schematically in FIG. 10.

A chimeric Notch receptor of the present disclosure can also be used tosimilarly modulate the activity of any other natural, chimeric, ororthogonal receptor whose activity is not constitutively present in thecell or not normally present in the cell.

In some cases, the cell is genetically modified to produce two or morechimeric Notch receptor polypeptides of the present disclosure. Thus, insome cases, the present disclosure provides a method of modulating anactivity of a cell that is genetically modified with: a) a nucleic acidcomprising a nucleotide sequence encoding two or more (e.g., 2, 3, 4, ormore) chimeric Notch polypeptide of the present disclosure; and b) anucleic acid comprising a nucleotide sequence encoding a chimericantigen receptor, where the nucleotide sequence encoding the chimericantigen receptor is under control of an inducible promoter. The methodinvolves: i) contacting the cell, which expresses the two or morechimeric Notch receptor polypeptides of the present disclosure (butwhich does not express the CAR), with a second member of the specificbinding pair (where the second member of the specific binding pair bindsto the first member, present in the chimeric Notch receptor polypeptide,of the specific binding pair), where contacting the cell with the secondmember of the specific binding pair induces release of the intracellulardomain of at least one of the two or more chimeric Notch receptorpolypeptide, where the intracellular domain is a transcription factorthat activates transcription of the nucleic acid comprising a nucleotidesequence encoding a chimeric antigen receptor, resulting in expressionof the CAR; and ii) after the contacting step of (i), contacting thecell with the antigen to which the CAR binds. The second contacting stepresults in modulation of activity by the CAR. In these embodiments,modulation of activity of the cell requires both a second member of thespecific binding pair, and the antigen to which the CAR specificallybinds.

A chimeric Notch receptor of the present disclosure can also be used tosimilarly modulate the activity of any other natural, chimeric, ororthogonal receptor whose activity is not constitutively present in thecell or not normally present in the cell. Multiple chimeric Notchreceptor polypeptides that function together can similarly be used tomodulate the activity of any other natural, chimeric, or orthogonalreceptor whose activity is not constitutively present in the cell or notnormally present in the cell.

Combinatorial Use of Chimeric Notch Receptor Polypeptides—MultipleReceptors in Series; Two Different Chimeric Notch Polypeptides

The present disclosure provides a method of modulating an activity of acell that is genetically modified with: i) a nucleic acid comprising anucleotide sequence encoding a first chimeric Notch polypeptide of thepresent disclosure; and b) a nucleic acid comprising a nucleotidesequence encoding a second chimeric Notch polypeptide of the presentdisclosure. For example, in some cases, the cell is genetically modifiedwith: i) a nucleic acid comprising a nucleotide sequence encoding afirst chimeric Notch receptor polypeptide comprising a first member of afirst specific binding pair; and ii) a nucleic acid comprising anucleotide sequence encoding at least a second chimeric Notch receptorpolypeptide comprising a first member of a second specific binding pair,where the first and the second specific binding pairs are different fromone another such that binding of a second member of the first specificbinding pair to the first member of the first specific binding pair doesnot result in release of the intracellular domain of the second chimericNotch receptor polypeptide, and such that binding of a second member ofthe second specific binding pair to the first member of the secondspecific binding pair does not result in release of the intracellulardomain of the first second chimeric Notch receptor polypeptide. In theseembodiments, the intracellular domain of the first chimeric Notchreceptor polypeptide provides a first effector function, where theeffector function provides for induction of transcription of the secondchimeric Notch polypeptide; and the intracellular domain of the secondchimeric Notch receptor polypeptide provides a second effector functionthat is different from the first effector function.

The method involves: i) contacting the cell, which expresses the firstchimeric Notch receptor polypeptide (but which does not express thesecond chimeric Notch receptor polypeptide), with a second member of thefirst specific binding pair (where the second member of the firstspecific binding pair binds to the first member, present in the firstchimeric Notch receptor polypeptide, of the first specific bindingpair), where contacting the cell with the second member of the firstspecific binding pair induces release of the intracellular domain of thefirst chimeric Notch receptor polypeptide, where the intracellulardomain of the first chimeric Notch receptor polypeptide is atranscription factor that activates transcription of the nucleic acidcomprising a nucleotide sequence encoding the second chimeric Notchreceptor polypeptide, resulting in expression of the second chimericNotch receptor polypeptide; and ii) after the contacting step of (i),contacting the cell with the second member of the second specificbinding pair (where the second member of the second specific bindingpair binds to the first member, present in the second chimeric Notchreceptor polypeptide, of the second specific binding pair). The secondcontacting step results in release of the intracellular domain of thesecond chimeric Notch receptor polypeptide, where the intracellulardomain of the second chimeric Notch receptor polypeptide provides aneffector function that modulates activity of the cell. In theseembodiments, modulation of activity of the cell requires contacting thecell first with a second member of the first specific binding pair, andthen with the second member of the second specific binding pair. Anexample of these embodiments is illustrated schematically in FIG. 11.

Two or more chimeric Notch receptors of the present disclosure can alsobe used in series in this manner to similarly modulate the activity ofany other natural, chimeric, or orthogonal receptor whose activity isnot constitutively present in the cell or not normally present in thecell.

Methods Involving Multiple Receptor Circuits with Two or More Cells

The present disclosure provides a method for modulating the activity ofa first cell, the method comprising contacting the first cell with asecond cell, where the second cell expresses a chimeric Notch receptorpolypeptide comprising an extracellular domain comprising a first memberof a specific binding pair; and where the second cell expresses on itssurface a molecule comprising the second member of the specific bindingpair. In some cases, contacting of the first cell with the second cellmodulates an activity in the first cell. In some cases, contacting ofthe first cell with the second cell modulates an activity in the secondcell.

The present disclosure provides a method of modulating the activity of afirst cell, the method involving contacting the first cell with a secondcell, where the second cell expresses a first chimeric Notch receptorpolypeptide comprising an extracellular domain comprising a first memberof a first specific binding pair; and the first cell expresses a secondchimeric Notch receptor polypeptide comprising an extracellular domaincomprising a first member of a second specific binding pair, and wherethe first cell comprises a nucleic acid comprising a nucleotide sequenceencoding a CAR. The second cell is contacted with a second member of thefirst specific binding pair, resulting in release of the intracellulardomain of the first chimeric Notch receptor polypeptide, where theintracellular domain of the first chimeric Notch receptor polypeptide isa transcription factor that induces transcription of a nucleic acidencoding the second member of the second specific binding pair. Thesecond member of the second specific binding pair is expressed on thesurface of the second cell. When the second member of the secondspecific binding pair, expressed on the surface of the second cell,comes into contact with the first cell, the second member of the secondspecific binding pair binds to the first member present in the secondchimeric Notch receptor polypeptide present on the cell surface of thefirst cell, resulting in release of the intracellular domain of thesecond chimeric Notch receptor polypeptide. The intracellular domain ofthe second chimeric Notch receptor polypeptide can be, e.g., atranscription factor that induces transcription of all or a part of aCAR, such that the CAR is expressed on the surface of the first cell. Insome cases, the first chimeric Notch receptor polypeptide present on thesurface of the second cell, and the CAR present on the surface of thefirst cell, recognize two separate antigens present on the surface of athird cell. In some cases, the third cell is a target cell. In somecases, the second cell is a “helper cell” that, when contacted with thefirst antigen, results in expression of the CAR on the surface of thefirst cell, where the CAR recognizes the second antigen on the targetcell. These embodiments are illustrated schematically in FIG. 12.

Methods of Modulating Cell Activity in a Multicellular Environment

A method of the present disclosure for modulating activity of a cell canbe carried out in a multicellular environment. For example, a firstchimeric Notch receptor polypeptide, present in a first cell (“firstreceiver cell”), can comprise an intracellular domain that, whenreleased after binding to a first ligand on a neighboring cell (e.g., a“sender” cell), provides for transcription of a second ligand to which asecond chimeric Notch receptor polypeptide on a second cell binds, wherethe second ligand is expressed on the surface of the first cell. Thesecond cell (“second receiver cell”) expresses the second chimeric Notchreceptor polypeptide on its surface; upon interaction with the firstreceiver cell, an intracellular effector function in the second receivercell is released an provides for modulation of the activity of thesecond receiver cell. These embodiments are illustrated schematically inFIG. 13. Such a method is useful for, e.g., constructing organizedtissues; tracking cell connectivities; and the like.

Methods of Modulating Cell Activity Involving Dual Recognition of aTarget Cell

In some cases, a method of the present disclosure for modulatingactivity of a cell involves recognition of two separate target moleculeson a target cell. For example, a first cell expressing a chimeric Notchreceptor polypeptide of the present disclosure recognizes a first targetmolecule on a target cell; and a second cell expressing on its cellsurface a receptor or other recognition molecule that binds a secondtarget molecule on the target cell. Binding of the first and secondcells to the first and second target molecules on the target cellresults in expression of a new effector function (e.g., a new geneproduct, such as a new polypeptide) by the second cell. In some cases,binding of the first cell to the first target molecule on the targetcell induces expression in the first cell of a gene product (e.g., apolypeptide); where in some cases, the new gene product produced by thefirst cell is expressed on the surface of the first cell and binds to areceptor or other recognition molecule on the surface of the secondcell, which binding may result modulation of an activity of the secondcell (e.g., induction of transcription of a nucleic acid such that thesecond cell expresses a new effector function (e.g., a new gene product,such as a new polypeptide). In some cases, binding of the first andsecond cells to the first and second target molecules on the target cellresults in killing of the target cell by the second cell, or killing ofthe target cell by the new effector function expressed by the secondcell. In some cases, binding of the first and second cells to the firstand second target molecules on the target cell results in modulation ofan activity of the target cell; e.g., where modulation of the activityof the target cell is induced by the new effector function produced bythe second cell. These embodiments are illustrated schematically in FIG.14.

Methods

The present disclosure provides a method of modulating an activity of acell that expresses binding-triggered transcriptional switch (e.g., achimeric Notch polypeptide of the present disclosure; a MESApolypeptide; a TANGO polypeptide; and the like). Methods of the presentdisclosure for modulating the activity of a cell can be carried out invitro, ex vivo, or in vivo. Methods of the present disclosure formodulating the activity of a cell can be carried out in a single cell,or in a multicellular environment (e.g., a naturally-occurring tissue;an artificial tissue; etc.). Methods of the present disclosure formodulating the activity of a cell can be carried out in parallel or inseries.

The present disclosure provides a method of locally modulating anactivity of a cell. The method generally involves: a) expressing in thecell a binding-triggered transcriptional switch comprising anextracellular domain comprising a first member of a specific bindingpair, a binding-transducer and an intracellular domain; and b)contacting the cell with a second member of the specific binding pair.Binding of the first member of the specific binding pair to the secondmember of the specific binding pair induces the binding-transducer totransduce a binding signal to activate the intracellular domain, therebyproducing an activated intracellular domain. The activated intracellulardomain modulates an activity of the cell. Activities of the cell thatcan be modulating using the method include, but are not limited to, i)expression of a gene product of the cell; ii) proliferation of the cell;iii) apoptosis of the cell; iv) non-apoptotic death of the cell; v)differentiation of the cell; vi) dedifferentiation of the cell; vii)migration of the cell; viii) secretion of a molecule from the cell; andix) cellular adhesion of the cell. In some cases, the contacting step iscarried out in vivo. In some cases, the contacting step is carried outex vivo. In some cases, the contacting step is carried out in vitro.

The present disclosure provides a method of locally modulating anactivity of a cell. The method generally involves: a) expressing in thecell a chimeric Notch polypeptide of the present disclosure, where thechimeric Notch polypeptide comprises: i) an extracellular domaincomprising a first member of a specific binding pair; ii) a Notchreceptor polypeptide, where the Notch receptor polypeptide is asdescribed above, and comprises one or more ligand-inducible proteolyticcleavage sites; and iii) an intracellular domain; and b) contacting thecell with a second member of the specific binding pair. Binding of thefirst member of the specific binding pair to a second member of thespecific binding pair induces cleavage of the Notch receptor polypeptideat the one or more ligand-inducible proteolytic cleavage sites, therebyreleasing the intracellular domain. The released intracellular domainmodulates an activity of the cell. Activities of the cell that can bemodulating using the method include, but are not limited to, i)expression of a gene product of the cell; ii) proliferation of the cell;iii) apoptosis of the cell; iv) non-apoptotic death of the cell; v)differentiation of the cell; vi) dedifferentiation of the cell; vii)migration of the cell; viii) secretion of a molecule from the cell; andix) cellular adhesion of the cell. In some cases, the contacting step iscarried out in vivo. In some cases, the contacting step is carried outex vivo. In some cases, the contacting step is carried out in vitro.

The present disclosure provides a method of locally modulating anactivity of a cell. The method generally involves: a) expressing in thecell a MESA polypeptide comprising an extracellular domain comprising afirst member of a specific binding pair, a binding-transducer and anintracellular domain; and b) contacting the cell with a second member ofthe specific binding pair. Binding of the first member of the specificbinding pair to the second member of the specific binding pair inducesthe binding-transducer to transduce a binding signal to release theintracellular domain, thereby producing a released intracellular domain.The released intracellular domain modulates an activity of the cell.Activities of the cell that can be modulating using the method include,but are not limited to, i) expression of a gene product of the cell; ii)proliferation of the cell; iii) apoptosis of the cell; iv) non-apoptoticdeath of the cell; v) differentiation of the cell; vi) dedifferentiationof the cell; vii) migration of the cell; viii) secretion of a moleculefrom the cell; and ix) cellular adhesion of the cell. In some cases, thecontacting step is carried out in vivo. In some cases, the contactingstep is carried out ex vivo. In some cases, the contacting step iscarried out in vitro.

The present disclosure provides a method of locally modulating anactivity of a cell. The method generally involves: a) expressing in thecell a TANGO polypeptide comprising an extracellular domain comprising afirst member of a specific binding pair, a binding-transducer and anintracellular domain; and b) contacting the cell with a second member ofthe specific binding pair. Binding of the first member of the specificbinding pair to the second member of the specific binding pair inducesthe binding-transducer to transduce a binding signal to release theintracellular domain, thereby producing a released intracellular domain.The released intracellular domain modulates an activity of the cell.Activities of the cell that can be modulating using the method include,but are not limited to, i) expression of a gene product of the cell; ii)proliferation of the cell; iii) apoptosis of the cell; iv) non-apoptoticdeath of the cell; v) differentiation of the cell; vi) dedifferentiationof the cell; vii) migration of the cell; viii) secretion of a moleculefrom the cell; and ix) cellular adhesion of the cell. In some cases, thecontacting step is carried out in vivo. In some cases, the contactingstep is carried out ex vivo. In some cases, the contacting step iscarried out in vitro.

In some cases, the activated (or released) intracellular domainmodulates expression of an endogenous gene product of the cell. In somecases, the endogenous gene product of the cell is a chemokine, achemokine receptor, a cytokine, a cytokine receptor, a differentiationfactor, a growth factor, a growth factor receptor, a hormone, ametabolic enzyme, a proliferation inducer, a receptor, a small moleculesecond messenger synthesis enzyme, a T cell receptor, a transcriptionactivator, a transcription repressor, a transcriptional activator, atranscriptional repressor, a translation regulator, a translationalactivator, a translational repressor, an activating immunoreceptor, anapoptosis in inhibitor, an apoptosis inducer, an immunoactivator, animmunoinhibitor, or an inhibiting immunoreceptor. In some cases, theendogenous gene product is a secreted gene product. In some cases, theendogenous gene product is a cell surface gene product. In some cases,the endogenous gene product is an intracellular gene product. In somecases, the activated intracellular domain simultaneously modulatesexpression of two or more endogenous gene products in the cell. An“activated intracellular domain” can be a released intracellular domain(e.g., released by proteolytic cleavage of the binding-triggeredtranscriptional switch. An “activated intracellular domain” can be aphosphorylated intracellular domain, e.g., where the intracellulardomain is inactive in its non-phosphorylated state, and active in itsphosphorylated state.

In some cases, the activated (or released) intracellular domainmodulates expression of a heterologous gene product in the cell. Aheterologous gene product is one that is not normally produced by thecell. For example, the cell can be genetically modified with a nucleicacid comprising a nucleotide sequence encoding the heterologous geneproduct. In some cases, the heterologous gene product is a chemokine, achemokine receptor, a chimeric antigen receptor, a cytokine, a cytokinereceptor, a differentiation factor, a growth factor, a growth factorreceptor, a hormone, a metabolic enzyme, a pathogen derived protein, aproliferation inducer, a receptor, a RNA guided nuclease, asite-specific nuclease, a small molecule second messenger synthesisenzyme, a T cell receptor, a toxin derived protein, a transcriptionactivator, a transcription repressor, a transcriptional activator, atranscriptional repressor, a translation regulator, a translationalactivator, a translational repressor, an activating immunoreceptor, anantibody, an apoptosis in inhibitor, an apoptosis inducer, an engineeredT cell receptor, an immunoactivator, an immunoinhibitor, an inhibitingimmunoreceptor, an RNA guided DNA binding protein, a synNotchpolypeptide of the present disclosure, a MESA polypeptide, a TANGOpolypeptide, a CAR, a TCR, or a second binding-triggered transcriptionalswitch. In some cases, the heterologous gene product is a secreted geneproduct. In some cases, the heterologous gene product is a cell surfacegene product. In some cases, the heterologous gene product is anintracellular gene product. In some cases, the activated intracellulardomain simultaneously modulates expression of two or more heterologousgene products in the cell. An “activated intracellular domain” can be areleased intracellular domain (e.g., released by proteolytic cleavage ofthe binding-triggered transcriptional switch. An “activatedintracellular domain” can be a phosphorylated intracellular domain,e.g., where the intracellular domain is inactive in itsnon-phosphorylated state, and active in its phosphorylated state.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a synNotch polypeptide of the presentdisclosure. In some cases, the released intracellular domain is anintracellular domain of a first synNotch polypeptide of the presentdisclosure, where the released intracellular domain of the firstsynNotch polypeptide induces expression of a second synNotch polypeptideof the present disclosure.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a CAR. In some cases, releasedintracellular domain is an intracellular domain of a synNotchpolypeptide of the present disclosure, where the released intracellulardomain of the synNotch polypeptide induces expression of a CAR.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a MESA polypeptide. In some cases, releasedintracellular domain is an intracellular domain of a synNotchpolypeptide of the present disclosure, where the released intracellulardomain of the synNotch polypeptide induces expression of a MESApolypeptide.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a TANGO polypeptide. In some cases,released intracellular domain is an intracellular domain of a synNotchpolypeptide of the present disclosure, where the released intracellulardomain of the synNotch polypeptide induces expression of a TANGOpolypeptide.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a TCR. In some cases, releasedintracellular domain is an intracellular domain of a synNotchpolypeptide of the present disclosure, where the released intracellulardomain of the synNotch polypeptide induces expression of a TCR.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a MESA polypeptide. In some cases, thereleased intracellular domain is an intracellular domain of a first MESApolypeptide, where the released intracellular domain of the first MESApolypeptide induces expression of a second MESA polypeptide.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a CAR. In some cases, releasedintracellular domain is an intracellular domain of a MESA polypeptide,where the released intracellular domain of the MESA polypeptide inducesexpression of a CAR.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a synNotch polypeptide of the presentdisclosure. In some cases, released intracellular domain is anintracellular domain of a MESA polypeptide, where the releasedintracellular domain of the MESA polypeptide induces expression of asynNotch polypeptide of the present disclosure.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a TANGO polypeptide. In some cases,released intracellular domain is an intracellular domain of a MESApolypeptide, where the released intracellular domain of the MESApolypeptide induces expression of a TANGO polypeptide.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a TCR. In some cases, releasedintracellular domain is an intracellular domain of a MESA polypeptide,where the released intracellular domain of the MESA polypeptide inducesexpression of a TCR.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a TANGO polypeptide. In some cases, thereleased intracellular domain is an intracellular domain of a firstTANGO polypeptide, where the released intracellular domain of the firstTANGO polypeptide induces expression of a second TANGO polypeptide.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a CAR. In some cases, releasedintracellular domain is an intracellular domain of a TANGO polypeptide,where the released intracellular domain of the TANGO polypeptide inducesexpression of a CAR.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a synNotch polypeptide of the presentdisclosure. In some cases, released intracellular domain is anintracellular domain of a TANGO polypeptide, where the releasedintracellular domain of the TANGO polypeptide induces expression of asynNotch polypeptide of the present disclosure.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a MESA polypeptide. In some cases, releasedintracellular domain is an intracellular domain of a TANGO polypeptide,where the released intracellular domain of the TANGO polypeptide inducesexpression of a MESA polypeptide.

In some cases, the activated (e.g., released) intracellular domaininduces expression of a heterologous gene product in the cell, where theheterologous gene product is a TCR. In some cases, releasedintracellular domain is an intracellular domain of a TANGO polypeptide,where the released intracellular domain of the TANGO polypeptide inducesexpression of a TCR.

In any of the above-described embodiments, the second member of thespecific binding pair can be on the surface of a second cell, can beimmobilized on an insoluble substrate, can be present in anextracellular matrix, can be present in an artificial matrix, or can besoluble.

MESA

A modular extracellular sensor architecture (MESA) polypeptide suitablefor use in a method of the present disclosure can be a MESA polypeptideas described in U.S. Patent Publication No. 2014/0234851. A MESApolypeptide comprises: a) a ligand binding domain; b) a transmembranedomain; c) a protease cleavage site; and d) a functional domain. Thefunctional domain can be a transcription regulator (e.g., atranscription activator, a transcription repressor). In some cases, aMESA receptor comprises two polypeptide chains. In some cases, a MESAreceptor comprises a single polypeptide chain.

TANGO

A suitable TANGO polypeptide is a heterodimer in which a first comprisesa tobacco etch virus (Tev) protease and a second polypeptide comprises aTev proteolytic cleavage site (PCS) fused to a transcription factor.When the two polypeptides are in proximity to one another, whichproximity is mediated by a native protein-protein interaction, Tevcleaves the PCS to release the transcription factor. Barnea et al. (ProcNatl Acad Sci USA. 2008 Jan. 8; 105(1):64-9).

TCR

In some cases, a binding-triggered switch induces expression of a T-cellreceptor (TCR) in a cell. TCR that can be induced using a method of thepresent disclosure include TCR that are specific for any of a variety ofepitopes, including, e.g., an epitope on the surface of a cancer cell,an epitope on the surface of a virus-infected cell, an epitope presentin an autoantigen, and the like. A TCR generally includes an alpha chainand a beta chain; and recognizes antigen when presented by a majorhistocompatibility complex. In some cases, the TCR is an engineered TCR.

Any engineered TCR having immune cell activation function can be inducedusing a method of the present disclosure. Such TCRs include, e.g.,antigen-specific TCRs, Monoclonal TCRs (MTCRs), Single chain MTCRs, HighAffinity CDR2 Mutant TCRs, CD1-binding MTCRs, High Affinity NY-ESO TCRs,VYG HLA-A24 Telomerase TCRs, including e.g., those described in PCT PubNos. WO 2003/020763, WO 2004/033685, WO 2004/044004, WO 2005/114215, WO2006/000830, WO 2008/038002, WO 2008/039818, WO 2004/074322, WO2005/113595, WO 2006/125962; Strommes et al. Immunol Rev. 2014;257(1):145-64; Schmitt et al. Blood. 2013; 122(3):348-56; Chapuls et al.Sci Transl Med. 2013; 5(174):174ra27; Thaxton et al. Hum VaccinImmunother. 2014; 10(11):3313-21 (PMID:25483644); Gschweng et al.Immunol Rev. 2014; 257(1):237-49 (PMID:24329801); Hinrichs et al.Immunol Rev. 2014; 257(1):56-71 (PMID:24329789); Zoete et al. FrontImmunol. 2013; 4:268 (PMID:24062738); Marr et al. Clin Exp Immunol.2012; 167(2):216-25 (PMID:22235997); Zhang et al. Adv Drug Deliv Rev.2012; 64(8):756-62 (PMID:22178904); Chhabra et al. Scientific WorldJournal. 2011; 11:121-9 (PMID:21218269); Boulter et al. Clin ExpImmunol. 2005; 142(3):454-60 (PMID:16297157); Sami et al. Protein EngDes Sel. 2007; 20(8):397-403; Boulter et al. Protein Eng. 2003;16(9):707-11; Ashfield et al. IDrugs. 2006; 9(8):554-9; Li et al. NatBiotechnol. 2005; 23(3):349-54; Dunn et al. Protein Sci. 2006;15(4):710-21; Liddy et al. Mol Biotechnol. 2010; 45(2); Liddy et al. NatMed. 2012; 18(6):980-7; Oates, et al. Oncoimmunology. 2013; 2(2):e22891;McCormack, et al. Cancer Immunol Immunother. 2013 April; 62(4):773-85;Bossi et al. Cancer Immunol Immunother. 2014; 63(5):437-48 and Oates, etal. Mol Immunol. 2015 October; 67(2 Pt A):67-74; the disclosures ofwhich are incorporated herein by reference in their entirety.

CAR

In some cases, a binding-triggered switch induces expression of a CAR ina cell. The terms “chimeric antigen receptor” and “CAR”, usedinterchangeably herein, refer to artificial multi-module moleculescapable of triggering or inhibiting the activation of an immune cellwhich generally but not exclusively comprise an extracellular domain(e.g., a ligand/antigen binding domain), a transmembrane domain and oneor more intracellular signaling domains. The term CAR is not limitedspecifically to CAR molecules but also includes CAR variants. CARvariants include split CARs wherein the extracellular portion (e.g., theligand binding portion) and the intracellular portion (e.g., theintracellular signaling portion) of a CAR are present on two separatemolecules. CAR variants also include ON-switch CARs which areconditionally activatable CARs, e.g., comprising a split CAR whereinconditional hetero-dimerization of the two portions of the split CAR ispharmacologically controlled. CAR variants also include bispecific CARs,which include a secondary CAR binding domain that can either amplify orinhibit the activity of a primary CAR. CAR variants also includeinhibitory chimeric antigen receptors (iCARs) which may, e.g., be usedas a component of a bispecific CAR system, where binding of a secondaryCAR binding domain results in inhibition of primary CAR activation. CARmolecules and derivatives thereof (i.e., CAR variants) are described,e.g., in PCT Application No. US2014/016527; Fedorov et al. Sci TranslMed (2013); 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21;Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al.Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33;Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu RevMed (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98;Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosuresof which are incorporated herein by reference in their entirety.

Spit CAR may be extracellularly split or intracellularly split and mayor may not be conditionally heterodimerizable. For example, split CARsystems that are not conditionally heterodimerizable may contain aconstitutive heterodimerization domain or other binding pair (e.g., a Fcbinding pair or other orthogonal binding pair) that does not depend onthe presence of one or more additional molecules for theheterodimerization that results in the formation of an active CAR fromassembly of the split portions.

In some instances, an extracellularly split CAR may be splitextracellularly at the antigen binding domain into two parts includinge.g., where the first part of the split CAR contains an extracellular Fcbinding domain that specifically binds to second part of the split CARthat contains the antigen recognition domain as generally depicted inFIG. 129A.

In some instances, an extracellularly split CAR may be splitextracellularly at the antigen binding domain into two parts includinge.g., where the first part of the split CAR contains an first part of anorthogonal protein binding pair that specifically binds to the secondpart of the orthogonal protein binding pair that is contained in thesecond part of the split CAR that contains the antigen recognitiondomain as generally depicted in FIG. 129B.

In some instances, an intracellularly split CAR may be splitintracellularly proximal to the transmembrane domain into two partsincluding e.g., where the first part of the split CAR includes theantigen recognition domain, a transmembrane domain and an intracellularfirst portion of a constitutive heterodimerization domain and the secondpart of the split CAR includes a transmembrane domain, the secondportion of the constitutive heterodimerization domain proximal to thetransmembrane domain, one or more co-stimulatory domains and one or moresignaling domains (e.g., ITAM domains) e.g., as generally depicted inFIG. 129C.

In some instances, an intracellularly split CAR may be split into twoparts intracellularly proximal to an intracellular domain or between twointracellular domains including e.g., where the first part of the splitCAR includes the antigen recognition domain, a transmembrane domain, oneor more co-stimulatory domains and an intracellular first portion of aconstitutive heterodimerization domain and the second part of the splitCAR includes a transmembrane domain, one or more co-stimulatory domains,one or more signaling domains (e.g., ITAM domains) and the secondportion of the constitutive heterodimerization domain between the one ormore co-stimulatory domains and the one or more signaling domains, e.g.,as generally depicted in FIG. 129D.

In some instances, an intracellularly split CAR may be split into twoparts intracellularly between intracellular domains including e.g.,where the first part of the split CAR includes the antigen recognitiondomain, a transmembrane domain, one or more co-stimulatory domains andan intracellular first portion of a constitutive heterodimerizationdomain proximal to the intracellular terminus of the first part of thesplit CAR and the second part of the split CAR includes a transmembranedomain, one or more signaling domains (e.g., ITAM domains) and thesecond portion of the constitutive heterodimerization domain between thetransmembrane domain and the one or more signaling domains, e.g., asgenerally depicted in FIG. 129E.

An ordinary skilled artisan will be readily aware that arrangements ofthe domains within first and second parts of a split CAR are not limitedto those arrangements specifically described herein. The specificlocations at which a single CAR may be split to generate a split CAR mayvary provided that the two or more polypeptides that result from such asplit or a plurality of splits are functionally capable of forming afunctional CAR upon their concurrent presence within a single cell. Suchfunctional activity may be readily determined including e.g., throughthe use of one or more of the assays described herein.

First Member of Specific Binding Pair

The first member of the specific binding pair can be the first member ofany of a variety of specific binding pairs. Suitable specific bindingpairs are described in detail above.

In some cases, the first member of the specific binding pair comprisesan antibody-based recognition scaffold. In some cases, the first memberof the specific binding pair comprises an antibody. In some cases, wherethe first member of the specific binding pair is an antibody, theantibody specifically binds a tumor-specific antigen, adisease-associated antigen, or an extracellular matrix component. Insome cases, where the first member of the specific binding pair is anantibody, the antibody specifically binds a cell surface antigen, asoluble antigen, or an antigen immobilized on an insoluble substrate. Insome cases, where the first member of the specific binding pair is anantibody, the antibody is a single-chain Fv. In some cases, the firstmember of the specific binding pair is a nanobody, a single-domainantibody, a diabody, a triabody, or a minibody. In some cases, the firstmember of the specific binding pair is a non-antibody-based recognitionscaffold. In some cases, where the first member of the specific bindingpair is a non-antibody-based recognition scaffold, thenon-antibody-based recognition scaffold is an avimer, a DARPin, anadnectin, an avimer, an affibody, an anticalin, or an affilin. In somecases, the first member of the specific binding pair is an antigen. Insome cases, where the first member of the specific binding pair is anantigen, the antigen is an endogenous antigen. In some cases, where thefirst member of the specific binding pair is an antigen, the antigen isan exogenous antigen. In some cases, the first member of the specificbinding pair is a ligand for a receptor. In some cases, the first memberof the specific binding pair is a receptor. In some cases, the firstmember of the specific binding pair is a cellular adhesion molecule(e.g., all or a portion of an extracellular region of a cellularadhesion molecule). In some cases, the first member of the specificbinding pair comprises a first dimerization domain and wherein thesecond member of the specific binding pair comprises a seconddimerization domain; for example, in some cases, binding of the firstdimerization domain to the second dimerization domain is induced by asmall molecule dimerization agent, and in other cases, binding of thefirst dimerization domain to the second dimerization domain is inducedby light.

Second Member of Specific Binding Pair

Specific binding pairs include, e.g., antigen-antibody specific bindingpairs, where the first member is an antibody (or antibody-basedrecognition scaffold) that binds specifically to the second member,which is an antigen, or where the first member is an antigen and thesecond member is an antibody (or antibody-based recognition scaffold)that binds specifically to the antigen; ligand-receptor specific bindingpairs, where the first member is a ligand and the second member is areceptor to which the ligand binds, or where the first member is areceptor, and the second member is a ligand that binds to the receptor;non-antibody-based recognition scaffold-target specific binding pairs,where the first member is a non-antibody-based recognition scaffold andthe second member is a target that binds to the non-antibody-basedrecognition scaffold, or where the first member is a target and thesecond member is a non-antibody-based recognition scaffold that binds tothe target; adhesion molecule-extracellular matrix binding pairs; Fcreceptor-Fc binding pairs, where the first member comprises animmunoglobulin Fc that binds to the second member, which is an Fcreceptor, or where the first member is an Fc receptor that binds to thesecond member which comprises an immunoglobulin Fc; andreceptor-co-receptor binding pairs, where the first member is a receptorthat binds specifically to the second member which is a co-receptor, orwhere the first member is a co-receptor that binds specifically to thesecond member which is a receptor.

The second member of the specific binding pair can be present on thesurface of a cell. The second member of the specific binding pair can beimmobilized on an insoluble support. The second member of the specificbinding pair can be soluble. The second member of the specific bindingpair can be present in an extracellular environment (e.g., extracellularmatrix). The second member of the specific binding pair can be presentin an artificial matrix. The second member of the specific binding paircan be present in an acellular environment.

Intracellular Domain

In some cases, the intracellular domain is a transcription regulator,e.g., a transcription factor such as a transcriptional activator or atranscriptional repressor. In some cases, the transcription factordirectly regulates differentiation of the cell. In some cases, thetranscription factor indirectly modulates differentiation of the cell bymodulating the expression of a second transcription factor.

Examples of transcriptional regulators include, e.g., ABT1, ACYP2,AEBP1, AEBP2, AES, AFFI, AFF3, AHR, ANKI, ANK2, ANKFY, ANKIBI, ANKRD1,ANKRD10, ANKRD2, ANKRD32, ANKRD46, ANKRD49, ANKRD56, ANKRD57, ANKS4B,AR, ARHGAP17, ARID1A, ARIDIB, ARID3A, ARID4A, ARID5B, ARNT, ARNT2,ARNTL, ARNTL2, ARX, ASB10, ASB11, ASB12, ASB15, ASB2, ASB5, ASB8, ASB9,ASH1L, ASH2L, ASXL1, ASZ1, ATF1, ATF3, ATF4, ATF4, ATF5, ATF6, ATF7,ATF7IP, ATM, ATOH1, ATXN3, 1300003B13RIK, B3GAT3, B930041F14RIK, BACH1,BACH2, BARX1, BARX2, BATF, BATF2, BATF3, BAZ2A, BBX, BC003267, BCL11A,BCL11B, BCL3, BCL6, BCL6B, BCLAF1, BCOR, BHLHA15, BHLHE40, BHLHE41,BLZF1, BMYC, BNC1, BNC2, BPNT1, BRCA1, BRWD1, BTBD11, BTF3,6030408C04RIK, CAMK4, CARHSP1, CARM1, CBX4, CBX7, CCNC, CCNH, CCNT1,CCNT2, CDCl₅L, CDK2, CDK4, CDK9, CDKN2C, CDX1, CDX1, CDX2, CEBPA, CEBPB,CEBPD, CEBPG, CEBPG, CEBPZ, CHD4, CHD7, CHGB, CIC, CIITA, CITEDI,CITED2, CITED4, CLOCK, CLPB, CML3, CNOT7, COPS2, CREB1, CREB3, CREB3L1,CREB3L1, CREB3L2, CREB3L3, CREB5, CREBBP, CREBL2, CREM, CSDA, CSDA,CSDC2, CSDE1, CTBP2, CTCF, CTCFL, CTNNB1, CTNNBL1, CXXC1, D11BWG0517E,2300002D11RIK, DACH1, DAXX, DBP, DDIT3, DDX20, DDX54, DDX58, DEAFI, DEK,DIDO1, DLX2, DMRT1, DMRT2, DMRTB1, DNMT1, DNMT3A, DR1, DRG1, DUSP26,DYSFIPI, E2F1, E2F2, E2F3, E2F5, E2F6, EBF1, EBF2, EBF3, EBF3, EED,EGRI, EGR2, EGR3, EHF, EHMT2, EID2, ELAVL2, ELFI, ELF1, ELF2, ELF3,ELF4, ELF5, ELK3, ELK4, ELL2, EMX2, EMX2, EN2, ENPP2, EOMES, EP300,EPAS1, ERF, ERG, ESR1, ESRRA, ESRRB, ESRRG, ETS1, ETS2, ETV1, ETV3,ETV4, ETV5, ETV6, EVIl, EWSR1, EZH1, EZH2, FAH, FBXL10, FBXLll, FBXW7,FEM1A, FEM1B, FEM1C, FHL2, FLII, FMNL2, FOS, FOSB, FOSL1, FOSL2, FOXA1,FOXA2, FOXA3, FOXC1, FOXD1, FOXD2, FOXD3, FOXF1, FOXF1A, FOXF2, FOXG1,FOXII, FOXJ2, FOXJ3, FOXK1, FOXK2, FOXL1, FOXL2, FOXM1, FOXN1, FOXN2,FOXN3, FOXO1, FOXO3, FOXPI, FOXP2, FOXP3, FOXP4, FOXQ1, FUS, FUSIPI,2810021G02RIK, GABPA, GABPB1, GARNL1, GAS7, GATA1, GATA2, GATA3, GATA4,GATA5, GATA5, GATA6, GBX2, GCDH, GCM1, GFI1, GFI1B, GLI2, GLI3, GLIS1,GLIS2, GLIS3, GLS2, GMEB1, GMEB2, GRHL1, GRHL2, GRHL3, GRLF1, GTF2A1,GTF2B, GTF2E2, GTF2F1, GTF2F2, GTF2H2, GTF2H4, GTF2I, GTF2IRD1,GTF2IRD1, GZF1, HAND2, HBP1, HCLS1, HDAC10, HDAC11, HDAC2, HDAC5, HDAC9,HELZ, HES1, HES4, HESS, HES6, HEXIMI, HEY2, HEYL, HHEX, HHEX, HIC1,HIC2, HIF1A, HIF1AN, HIPK2, HIVEPI, HIVEP2, HIVEP2, HIVEP3, HLF, HLTF,HLX, HMBOX1, HMG20A, HMGA2, HMGB2, HMGB3, HNF1B, HNF4A, HNF4G, HOMEZ,HOXA10, HOXA11, HOXA13, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9,HOXB1, HOXB2, HOXB3, HOXB4, HOXB6, HOXB7, HOXB8, HOXB9, HOXC10, HOXC10,HOXC11, HOXC5, HOXC6, HOXC8, HOXC9, HOXD8, HOXD9, HR, HSBP1, HSF2BP,HTATIP2, HTATSF1, HUWE1, 5830417I10RIK, ID1, ID2, ID3, ID3, IFNAR2,IKBKB, IKBKG, IKZF1, IKZF2, IKZF3, IKZF4, IL31RA, ILF3, INGI, ING2,ING3, ING4, INSM1, INTS12, IQWD1, IRFI, IRFI, IRF2, IRF3, IRF4, IRF5,IRF6, IRF7, IRF8, IRF8, IRX1, IRX2, IRX3, IRX4, IRX5, ISL1, ISL2, ISX,ISX, IVNS1ABP, 2810021J22RIK, JARID1A, JARIDIB, JARIDIC, JARIDID, JDP2,JUN, JUNB, JUND, KLF1, KLF10, KLF11, KLF12, KLF13, KLF15, KLF16, KLF2,KLF3, KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF9, KRR1, 6330416L07RIK,L3MBTL2, LASS2, LASS4, LASS6, LBA1, LBH, LBX1, LCOR, LDB1, LDB2, LEF1,LHX1, LHX2, LHX5, LIMD1, LIN28, LMO1, LMO4, LMX1A, LSM11, LSM4, LYL1,9030612M13RIK, 1810007M14RIK, 3632451O06RIK, MAF, MAFA, MAFB, MAFF,MAFG, MAFK, MAGEDI, MAP3K12, MAPK1, MAPK3, MAPK8, MAPK8IP1, MAX, MAZ,MBD2, MCM2, MCM4, MCM5, MCM6, MCM7, MECOM, MECP2, MED12, MED8, MEF2A,MEF2B, MEF2C, MEF2D, MEIS1, MEIS1, MEIS2, MEOX2, MESP2, MID1, MITF,MKI67IP, MKL1, MLL1, MLL3, MLLT10, MLLT3, MLX, MLXIP, MLXIPL, MNT, MNX1,MPL, MSC, MSRB2, MSX2, MTA3, MTF1, MTF2, MTPN, MXD1, MXD4, MXI1, MYB,MYBBP1A, MYBL2, MYC, MYCBP, MYCL1, MYCN, MYEF2, MYF6, MYNN, MYOCD,MYOD1, MYOG, MYST3, MYST4, MYT1L, MZF1, NAB1, NAB2, NANOG, NARGI, NCOA1,NCOA2, NCOA3, NCOR1, NCOR2, NDN, NEURODI, NEUROD4, NEUROD6, NEUROGI,NEUROG2, NFAT5, NFATC1, NFATC2, NFATC2IP, NFATC3, NFATC3, NFATC4, NFE2,NFE2L1, NFE2L2, NFIA, NFIA, NFIB, NFIC, NFIL3, NFIX, NFKB1, NFKB2,NFKBIB, NFKBIE, NFKBIZ, NFX1, NFXL1, NFYA, NFYB, NHLH1, NKX2-2, NKX2-3,NKX2-5, NKX2-6, NKX6-2, NMI, NOTCHI, NOTCH2, NOTCH3, NOTCH4, NPAS1,NPAS2, NPAS3, NROB1, NROB2, NR1D1, NR1D2, NR1H3, NR1H4, NR1I2, NR1I3,NR2C1, NR2C2, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2,NR4A2, NR4A3, NR5A1, NR5A2, NRARP, NRIP1, NRIP2, NSBP1, NSD1, NUDT12,NULL, NUPR1, 1700065013RIK, OLIGI, OLIG2, OLIG2, ONECUTI, ONECUT2,ONECUT3, ORC2L, OSGINI, OSRI, OSR2, OSTF1, OVOL1, OVOL2, PAPOLA, PAPOLG,PAPPA2, PATZ1, PAWR, PAX2, PAX5, PAX6, PAX7, PAX8, PAX9, PBX1, PBX2,PBX3, PBX4, PCBD1, PCGF6, PDCD11, PDLIM4, PDX1, PEG3, PERI, PFDN1, PGR,PHF1, PHF10, PHF12, PHF13, PHF14, PHF20, PHF21A, PHF5A, PHF7, PHOX2A,PHOX2B, PIAS2, PIR, PITX1, PITX2, PKNOX1, PKNOX2, PLA2G6, PLAGLI,PLAGL2, PLRG1, PML, POGK, POLR2B, POLR2E, POLR2H, POLR3E, POLR3H,POLRMT, POU1F1, POU2AF1, POU2F1, POU2F2, POU3F2, POU3F3, POU3F3, POU5F1,POU6F1, PPARA, PPARD, PPARG, PPARGC1A, PPARGClB, PPP1R12C, PPP1R13B,PPP1R16B, PPP1R1B, PPP2R1A, PPP3CB, PQBP1, PRDM1, PRDM14, PRDM15,PRDM16, PRDM2, PRDM4, PRDM5, PRDM6, PRDM8, PREB, PRKAR1A, PRKCBP1,PROX1, PRRX1, PRRX2, PSMC5, PSMD10, PSMD9, PTF1A, PTGES2, PURB, PWP1,RAB11A, RAB11B, RAB15, RAB18, RAB1B, RAB25, RAB8A, RAB8B, RAI14, RARA,RARB, RARG, RASSF7, RB1, RBBP7, RBL1, RBM14, RBM39, RBM9, RBPJ, RBPJL,RCOR2, REL, RELA, RELB, RERE, REST, REXO4, RFC1, RFX1, RFX2, RFX3, RFX5,RFX7, RFX8, RHOX5, RHOX6, RHOX9, RIPK4, RNF12, RNF14, RNF141, RNF38,RNF4, RORA, RORA, RORB, RORC, RPS6KA4, RREB1, RSRC1, RUNX1, RUNX1T1,RUNX2, RUNX2, RUNX3, RUVBL1, RUVBL2, RXRA, RXRG, RYBP, SAFB2, SALL1,SALL1, SALL2, SALL4, SAP30, SAP30BP, SATB1, SATB2, SATB2, SCANDI, SCAP,SCRT2, SEC14L2, SERTADI, SF1, SFPIl, SFRS5, SH3D19, SH3PXD2B, SHANK3,SHOX2, SHPRH, SIN3A, SIN3B, SIRT2, SIRT3, SIRT5, SIX1, SIX1, SIX2, SIX3,SIX4, SIX5, SKI, SMAD1, SMAD2, SMAD3, SMAD7, SMARCA1, SMARCA2, SMARCA5,SMARCB1, SMYD1, SNAIl, SNAI2, SNAPC2, SNAPC4, SNIPI, SOLH, SOX1, SOX10,SOX11, SOX12, SOX13, SOX15, SOX17, SOX18, SOX2, SOX21, SOX4, SOX5, SOX6,SOX7, SOX8, SOX9, SP1, SP110, SP140L, SP2, SP3, SP4, SP6, SP8, SPDEF,SPEN, SPIl, SPIB, SQSTM1, SREBF1, SREBF2, SREBF2, SRF, SSBP2, SSBP3,SSBP4, SSRP1, ST18, STAG1, STAT1, STAT1, STAT2, STAT3, STAT4, STAT5A,STAT5B, STAT5B, STAT6, SUB1, SUZ12, TADA2L, TAF13, TAF5, TAF5L, TAF7,TAF9, TAL, TALl, TARDBP, TBPL1, TBR1, TBX1, TBX10, TBX15, TBX18, TBX2,TBX2, TBX20, TBX21, TBX3, TBX4, TBX5, TBX6, TCEA1, TCEA3, TCEAL1, TCEB3,TCERG1, TCF12, TCF15, TCF19, TCF20, TCF21, TCF21, TCF3, TCF4, TCF7,TCF7L2, TCFAP2A, TCFAP2B, TCFAP2C, TCFCP2L1, TCFE2A, TCFE3, TCFEB,TCFEC, TCFL5, TEADI, TEAD2, TEAD3, TEAD4, TEF, TFAP2A, TFAP2C, TFCP2L1,TFDP2, TFEB, TFEC, TGFB1I1, TGIF1, TGIF2, TGIF2LX, THRA, THRAP3, THRB,THRSP, TIAL1, TLE1, TLE6, TMEM131, TMPO, TNFAIP3, TOBI, TOX4, TP63,TRERFI, TRIB3, TRIM24, TRIM28, TRIM30, TRIP13, TRIP4, TRIP6, TRP53,TRP53BP1, TRP63, TRPS1, TRPS1, TSC22D1, TSC22D2, TSC22D3, TSC22D4,TSHZ1, TSHZ1, TSHZ3, TTRAP, TUB, TULP4, TWISTI, TWIST2, TYSND1, UBE2W,UBN1, UBP1, UBTF, UGP2, UHRF1, UHRF2, UNCX, USFI, USF2, UTFI, VDR,VEZF1, VGLL2, VSX1, WASL, WHSC1, WHSC2, WT1, WWP1, WWTR1, XBP1, YAF2,YY1, ZBED1, ZBED4, ZBTB1, ZBTB10, ZBTB16, ZBTB16, ZBTB17, ZBTB2, ZBTB20,ZBTB22, ZBTB25, ZBTB32, ZBTB38, ZBTB4, ZBTB43, ZBTB45, ZBTB47, ZBTB7A,ZBTB7B, ZBTB7C, ZCCHC8, ZDHHC13, ZDHHC16, ZDHHC21, ZDHHC5, ZDHHC6, ZEB2,ANK2ZEB2, ZFHX2, ZFHX3, ZFHX4, ZFP105, ZFP110, ZFP143, ZFP148, ZFP161,ZFP192, ZFP207, ZFP219, ZFP238, ZFP263, ZFP275, ZFP277, ZFP281, ZFP287,ZFP292, ZFP35, ZFP354C, ZFP36, ZFP36L1, ZFP386, ZFP407, ZFP42, ZFP423,ZFP426, ZFP445, ZFP451, ATF5ZFP451, ZFP467, ZFP52, ZFP57, ZFP592,ZFP593, ZFP597, ZFP612, ZFP637, ZFP64, ZFP647, ZFP748, ZFP810, ZFP9,ZFP91, ZFPM1, ZFPM2, ZFX, ZHX2, ZHX3, ZICI, ZIC2, ZIC3, ZIC4, ZIC5,ZKSCAN1, ZKSCAN3, ZMYND11, ZNF143, ZNF160, ZNF175, ZNF184, ZNF192,ZNF213, ZNF217, ZNF219, ZNF22, ZNF238, ZNF24, ZNF267, ZNF273, ZNF276,ZNF280D, ZNF281, ZNF292, ZNF311, ZNF331, ZNF335, ZNF337, ZNF33B, ZNF366,ZNF394, ZNF398, ZNF41, ZNF410, ZNF415, ZNF423, ZNF436, ZNF444, ZNF445,ZNF451, ZNF460, ZNF496, ZNF498, ZNF516, ZNF521, ZNF532, ZNF536, ZNF546,ZNF552, ZNF563, ZNF576, ZNF580, ZNF596, ZNF621, ZNF628, ZNF648, ZNF649,ZNF652, ZNF655, ZNF664, ZNF668, ZNF687, ZNF692, ZNF696, ZNF697, ZNF710,ZNF80, ZNF91, ZNF92, ZNRD1, ZSCAN10, ZSCAN16, ZSCAN20, ZSCAN21, ZXDC,and ZZZ3. Additional examples of transcriptional regulators are asdescribed above. Non-limiting examples of transcription factors(transcriptional activators; transcriptional repressors) are depicted inFIGS. 37-83. For example, a transcription factor can comprise an aminoacid sequence having at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence depicted in any one ofFIGS. 37-83.

In some cases, activation (e.g., phosphorylation; release) of theintracellular domain modulates expression of an endogenous gene of thecell through transcriptional regulation, chromatin regulation,translation, trafficking or post-translational processing. In somecases, activation (e.g., phosphorylation; release) of the intracellulardomain modulates cellular adhesion of the cell to a second cell or to anextracellular matrix.

Binding-Transducer

The binding-transducer can comprise a ligand-inducible proteolyticcleavage site, where binding of the first member of the specific bindingpair to the second member of the specific binding pair induces cleavageof the binding-transducer at the ligand-inducible proteolytic cleavesite, thereby transducing the binding signal and activating theintracellular domain by proteolytically releasing the intracellulardomain. Examples of proteolytic cleavage sites in a synNotch are asdescribed above.

As other examples, the proteolytic cleavage site can be, e.g., ametalloproteinase cleavage site, e.g., a cleavage site for a MMPselected from collagenase-1, -2, and -3 (MMP-1, -8, and -13), gelatinaseA and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, -10, and -11),matrilysin (MMP-7), and membrane metalloproteinases (MT1-MMP andMT2-MMP). For example, the cleavage sequence of MMP-9 is Pro-X-X-Hy(wherein, X represents an arbitrary residue; Hy, a hydrophobic residue),e.g., Pro-X-X-Hy-(Ser/Thr), e.g., Pro-Leu/Gln-Gly-Met-Thr-Ser (SEQ IDNO:101) or Pro-Leu/Gln-Gly-Met-Thr (SEQ ID NO:102). Another example of aprotease cleavage site is a plasminogen activator cleavage site, e.g., auPA or a tissue plasminogen activator (tPA) cleavage site. Anotherexample of a suitable protease cleavage site is a prolactin cleavagesite. Specific examples of cleavage sequences of uPA and tPA includesequences comprising Val-Gly-Arg. Another example of a protease cleavagesite that can be included in a proteolytically cleavable linker is atobacco etch virus (TEV) protease cleavage site, e.g., ENLYTQS (SEQ IDNO:103), where the protease cleaves between the glutamine and theserine. Another example of a protease cleavage site that can be includedin a proteolytically cleavable linker is an enterokinase cleavage site,e.g., DDDDK (SEQ ID NO:104), where cleavage occurs after the lysineresidue. Another example of a protease cleavage site that can beincluded in a proteolytically cleavable linker is a thrombin cleavagesite, e.g., LVPR (SEQ ID NO:105). Additional suitable linkers comprisingprotease cleavage sites include linkers comprising one or more of thefollowing amino acid sequences: LEVLFQGP (SEQ ID NO:106), cleaved byPreScission protease (a fusion protein comprising human rhinovirus 3Cprotease and glutathione-S-transferase; Walker et al. (1994) Biotechnol.12:601); a thrombin cleavage site, e.g., CGLVPAGSGP (SEQ ID NO:107);SLLKSRMVPNFN (SEQ ID NO:108) or SLLIARRMPNFN (SEQ ID NO:109), cleaved bycathepsin B; SKLVQASASGVN (SEQ ID NO:110) or SSYLKASDAPDN (SEQ IDNO:111), cleaved by an Epstein-Barr virus protease; RPKPQQFFGLMN (SEQ IDNO:112) cleaved by MMP-3 (stromelysin); SLRPLALWRSFN (SEQ ID NO:113)cleaved by MMP-7 (matrilysin); SPQGIAGQRNFN (SEQ ID NO:114) cleaved byMMP-9; DVDERDVRGFASFL SEQ ID NO: 115) cleaved by a thermolysin-like MMP;SLPLGLWAPNFN (SEQ ID NO:116) cleaved by matrix metalloproteinase2(MMP-2); SLLIFRSWANFN (SEQ ID NO:117) cleaved by cathespin L;SGVVIATVIVIT (SEQ ID NO:118) cleaved by cathepsin D; SLGPQGIWGQFN (SEQID NO:119) cleaved by matrix metalloproteinase 1(MMP-1); KKSPGRVVGGSV(SEQ ID NO:120) cleaved by urokinase-type plasminogen activator;PQGLLGAPGILG (SEQ ID NO:121) cleaved by membrane type 1matrixmetalloproteinase (MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT (SEQID NO:122) cleaved by stromelysin 3 (or MMP-11), thermolysin, fibroblastcollagenase and stromelysin-1; GPQGLAGQRGIV (SEQ ID NO:123) cleaved bymatrix metalloproteinase 13 (collagenase-3); GGSGQRGRKALE (SEQ IDNO:124) cleaved by tissue-type plasminogen activator (tPA); SLSALLSSDIFN(SEQ ID NO:125) cleaved by human prostate-specific antigen; SLPRFKIIGGFN(SEQ ID NO:126) cleaved by kallikrein (hK3); SLLGIAVPGNFN (SEQ IDNO:127) cleaved by neutrophil elastase; and FFKNIVTPRTPP (SEQ ID NO:128)cleaved by calpain (calcium activated neutral protease).

Cells

A method of the present disclosure can be used to modulate an activityof any eukaryotic cell. In some cases, the cell is in vivo. In somecases, the cell is ex vivo. In some cases, the cell is in vitro. In somecases, the cell is a mammalian cell. In some cases, the cell is a humancell. In some cases, the cell is a non-human primate cell. In somecases, the cell is rodent cell. In some cases, the cell is mouse cell.In some cases, the cell is a rat cell.

Suitable cells include retinal cells (e.g., Müller cells, ganglioncells, amacrine cells, horizontal cells, bipolar cells, andphotoreceptor cells including rods and cones, Müller glial cells, andretinal pigmented epithelium); neural cells (e.g., cells of thethalamus, sensory cortex, zona incerta (ZI), ventral tegmental area(VTA), prefontal cortex (PFC), nucleus accumbens (NAc), amygdala (BLA),substantia nigra, ventral pallidum, globus pallidus, dorsal striatum,ventral striatum, subthalamic nucleus, hippocampus, dentate gyrus,cingulate gyrus, entorhinal cortex, olfactory cortex, primary motorcortex, or cerebellum); liver cells; kidney cells; immune cells; cardiaccells; skeletal muscle cells; smooth muscle cells; lung cells; and thelike.

Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, aninduced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, asperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. afibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, aneuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell,etc.

Suitable cells include human embryonic stem cells, fetal cardiomyocytes,myofibroblasts, mesenchymal stem cells, autotransplated expandedcardiomyocytes, adipocytes, totipotent cells, pluripotent cells, bloodstem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymalcells, embryonic stem cells, parenchymal cells, epithelial cells,endothelial cells, mesothelial cells, fibroblasts, osteoblasts,chondrocytes, exogenous cells, endogenous cells, stem cells,hematopoietic stem cells, bone-marrow derived progenitor cells,myocardial cells, skeletal cells, fetal cells, undifferentiated cells,multi-potent progenitor cells, unipotent progenitor cells, monocytes,cardiac myoblasts, skeletal myoblasts, macrophages, capillaryendothelial cells, xenogenic cells, allogenic cells, and post-natal stemcells.

In some cases, the cell is an immune cell, a neuron, an epithelial cell,and endothelial cell, or a stem cell. In some cases, the immune cell isa T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell,or a macrophage. In some cases, the immune cell is a cytotoxic T cell.In some cases, the immune cell is a helper T cell. In some cases, theimmune cell is a regulatory T cell (Treg).

In some cases, the cell is a stem cell. In some cases, the cell is aninduced pluripotent stem cell. In some cases, the cell is a mesenchymalstem cell. In some cases, the cell is a hematopoietic stem cell. In somecases, the cell is an adult stem cell.

Suitable cells include bronchioalveolar stem cells (BASCs), bulgeepithelial stem cells (bESCs), corneal epithelial stem cells (CESCs),cardiac stem cells (CSCs), epidermal neural crest stem cells (eNCSCs),embryonic stem cells (ESCs), endothelial progenitor cells (EPCs),hepatic oval cells (HOCs), hematopoetic stem cells (HSCs), keratinocytestem cells (KSCs), mesenchymal stem cells (MSCs), neuronal stem cells(NSCs), pancreatic stem cells (PSCs), retinal stem cells (RSCs), andskin-derived precursors (SKPs)

In some cases, the stem cell is a hematopoietic stem cell (HSC), and thetranscription factor induces differentiation of the HSC to differentiateinto a red blood cell, a platelet, a lymphocyte, a monocyte, aneutrophil, a basophil, or an eosinophil. In some cases, the stem cellis a mesenchymal stem cell (MSC), and the transcription factor inducesdifferentiation of the MSC into a connective tissue cell such as a cellof the bone, cartilage, smooth muscle, tendon, ligament, stroma, marrow,dermis, or fat.

Kits

The present disclosure provides a kit for carrying out a method ofmodulating the activity of a cell.

In some cases, a subject kit comprises an expression vector comprising anucleotide sequence encoding a chimeric Notch receptor polypeptide ofthe present disclosure. In some cases, a subject kit comprises achimeric Notch receptor polypeptide of the present disclosure.

In some cases, a subject kit comprises a host cell that is geneticallymodified with a nucleic acid comprising a nucleotide sequence encoding achimeric Notch receptor polypeptide of the present disclosure. In somecases, a subject kit comprises a host cell that is genetically modifiedwith a recombinant expression vector comprising a nucleotide sequenceencoding a chimeric Notch receptor polypeptide of the presentdisclosure. Kit components can be in the same container, or in separatecontainers.

Any of the above-described kits can further include one or moreadditional reagents, where such additional reagents can be selectedfrom: a dilution buffer; a reconstitution solution; a wash buffer; acontrol reagent; a control expression vector; a negative controlpolypeptide (e.g., a chimeric Notch receptor polypeptide that lacks theone or more ligand-inducible proteolytic cleavage sites, such that, uponbinding of the first member of the specific binding to the second memberof the specific binding pair, the intracellular domain is not released);a positive control polypeptide; a reagent for in vitro production of thechimeric Notch receptor polypeptide, and the like.

In addition to above-mentioned components, a subject kit can furtherinclude instructions for using the components of the kit to practice thesubject methods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or subpackaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, flash drive, etc. In yet other embodiments, the actualinstructions are not present in the kit, but means for obtaining theinstructions from a remote source, e.g. via the internet, are provided.An example of this embodiment is a kit that includes a web address wherethe instructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1: Generation and Characterization of Chimeric Notch ReceptorPolypeptides Materials and Methods

Jurkat T cells were stably transduced via lentivirus with a Tet responseelement (TRE) anti-Mesothelin CAR-EGFP/pGK mCherry dual vector.Transduced cells constitutively expressed mCherry and induciblyexpressed the anti-Mesothelin CAR-EGFP fusion in the presence of the Tettransactivator (tTa). This stable line was then transduced with theanti-CD19 Notch tTA receptor. The resultant Jurkat cells were thenassayed via co-culturing with Chronic Myeloid Leukemia K562 cancer cellsexpressing the antigens CD19, Mesothelin, or both. The T cells wereassessed at 24 hrs by flow cytometry for expression of the EGFP taggedCAR and the activation marker, CD69. The co-culture supernatant was alsocollected and secretion of IL-2 was determined by ELISA.

Results Chimeric Notch Receptor Characterization

To show that the Chimeric Notch platform could be used to inducetranscription in receiver cells upon ligand binding, the followingreporter cell lines were generated: mouse L929 fibroblasts reporterlines were generated by transduction with a Tet response element(TRE)-->EGFP reporter/pGK mCherry dual vector (TRE reporter) or a Gal4UAS-->EGFP/pGK puromycin resistance dual vector (UAS reporter). TheChimeric Notch variants were transduced into the corresponding L929reporter cell line, to generate Chimeric Notch reporter cells. TheseChimeric Notch reporter cells were stimulated in parallel by twomethods. First, the Chimeric Notch expressing cells were exposed to animmobilized antibody that bound specifically to the extracellular domainof the receptor. Second, the cells were incubated with K562 cells orL929 fibroblasts expressing the cognate Chimeric Notch ligand. TheChimeric Notch expressing cells were assayed for EGFP fluorescence byflow cytometry to measure reporter activity. FIGS. 30-32 showrepresentative results for the Chimeric Notch with anti-CD19 (FIG.30A-B) and anti-mesothelin (FIG. 31A-B) in the TRE reporter line, andfor the Chimeric Notch anti-CD19 for the UAS reporter line (FIG. 32A-B).

To show that the Chimeric Notch platform can be used to represstranscription, another reporter line was generated that expressed EGFPdownstream of a composite SV40/UAS promoter (SV40/UAS reporter). FIG.33A These SV40/UAS reporter cell line express GFP at high levels. TheseSV40/UAS reporter cells were transduced with the anti-CD19 ChimericNotch in which the intracellular domain is a fusion of the Gal4DNA-binding domain with the transcriptional repressor domain KRAB. Asshown in FIG. 33B, untreated Chimeric Notch SV40/UAS reporter cellsdisplay high EGFP expression; in the presence of the ligand for theChimeric Notch receptor, the EGFP expression is down-regulated.

To show that a cascade of signaling relay can be built, a cascade ofmultiple Chimeric Notch polypeptides was designed in the following way.Cells A express the first ligand A (mesothelin). Cells B express theChimeric Notch with anti-mesothelin scFv as extracellular domain and tTAas intracellular domain; cells B also express the second ligand, CD19,and a blue fluorescent protein (BFP) under the control of a TREsequence. Cells C express the anti-CD19 Chimeric Notch with tTA asintracellular domain; moreover, cells C express GFP controlled by a TREsequence. FIG. 34 shows a time course of microscope pictures of onerepresentative aggregate of cells A+cells B+cells C. Cells B, activatedby the ligand expressed by cells A, induce expression of BFP and CD19 atday 1 of cultivation; CD19 from cells B in turn induces expression ofGFP in cells C starting from day 2.

Chimeric Notch Gates Chimeric Antigen Receptor Expression and T CellActivation to Cancer Cells

T cells engineered to express artificial T cell receptors known asChimeric Antigen receptors (CAR) are effective as therapeutics forcertain B cell cancers. However, a major concern with CAR T cell cancerimmunotherapy is off-target effects, where the therapeutic T cellsdestroy normal tissue leading to serious side effects and even death. Apotential strategy to mitigate such problems is for therapeutic T cellsto only express the CAR when in the tumor microenvironment providingmore localized T cell responses. To implement such a strategy, it wasreasoned that Chimeric Notch could be used in therapeutic T cells tofirst detect the tumor by binding a tumor-specific cell surface antigenand initiate expression of a CAR to a second tumor-specific antigen onlyin the tumor. Effectively, this provides both dual antigen control overT cell activity and a tumor-localized response. Proof of principal invitro data are presented in Jurkat cells.

Jurkat T cells that express the CD19scFv Chimeric Notch tTA equippedwith a TRE driving the expression of a Mesothelin scFv CAR-EGFP wereexposed to Mesothelin+ or CD19/Mesothelin+K562 cancer cells and CARexpression and T cell activation was assessed at 24 hours (FIG. 35A).Upregulation of the activation marker, CD69, and IL-2 secretion wereused as indicators of T cell activation. The Chimeric Notch engineered Tcells only expressed the CAR and activated when exposed to cancer cellsexpressing both antigens (FIG. 35B-C).

FIGS. 35A-C. Chimeric Notch Gates Chimeric Antigen Receptor Expressionand T cell Activation to Cancer Cells. (A) Schematic of two-antigencontrol of T cell activation by anti-CD19 Chimeric Notch tTA gatedexpression of the anti-Mesothelin CAR. (B) Flow cytometry analysisCAR-GFP expression (left panel) and CD69 levels (right panel) of JurkatT cells that express the CD19scFV Chimeric Notch tTA equipped with a TREdriving the expression of a Mesothelin scFv CAR-EGFP after exposure toK562 cancer cells positive for the indicated antigens. (C) IL-2 levelsin the co-culture supernatant from the same experiment described inpanel B.

Example 2: Gamma Secretase Protease Activity and Chimeric NotchSignaling

A “receiver” cell expressing a chimeric Notch polypeptide (comprising ananti-CD19 scFv as the extracellular polypeptide, and a tTAtranscriptional activator as the intracellular domain), and including apTet-GFP construct, as depicted in FIG. 30A and as described in Example1, was contacted with a “sender” cell expressing CD19 on its surface, inthe presence or absence of 1 mMN-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethylester (DAPT). DAPT is a gamma secretase inhibitor. The data areexpressed as GFP+“receiver” cells. As shown in FIGS. 84A and 84B, in thepresence of the sender cells and DAPT, GFP+“receiver” cells were atcontrol, untreated levels; in the absence of DAPT, contact with thesender cells robustly activated the chimeric Notch polypeptide andexpression of GFP.

Example 3: Synthetic Notch Receptors are a Modular Platform forEngineering Customized Cell Sensing and Response Behaviors

A Minimal Notch Transmembrane Region can be Combined with NovelExtracellular and Intracellular Domains to Construct Diverse ChimericReceptors.

The core of the Notch transduction pathway is in the transmembraneregion. This design can be the thought of as a platform for engineeringa series of receptors that detect a diverse array of membrane-presentedligands (FIG. 85A). On the intracellular side, the Notch intracellulardomain (NICD) can be replaced by orthogonal transcription factors, toreport on Notch signaling activation. On the extracellular, theendogenous Delta binding domain can be replaced by protein bindingdomains (e.g. FKBP, anti-GFP nanobody, etc.) and cleavage is inducedwhen the cognate binding partner is bound. Therefore, libraries ofreceptor molecules with different extracellular domains each coupledwith a different intracellular domain were generated (FIG. 85B). Asshown in FIG. 86A, the synthetic receptor response is activated bycell-cell contact with sender cells expressing the cognate ligand in acleavage-dependent manner. Results on different extracellular andintracellular domain combinations are provided in FIG. 87A.

The activation of the synthetic receptor response is reversible uponremoval of the ligand expressing cells, and the transcriptional responseat 24h can be activated by a pulse of stimulation as short as 1h (FIG.87B); moreover, the dose/response relationship between the ligand amountand the receptor activation is graded, replicating a feature ofendogenous Notch signaling (FIG. 87C). The amount of receptor expressedby the cells correlates linearly with the strength of induction of thereporter (FIG. 87D). For certain extracellular domains, suboptimalbackground activity or poor inducibility were observed. It wasdiscovered that, by slightly extending the regulated cleavagetransmembrane region of the synthetic Notch to incorporate one or moreEGF-repeats could sometimes improve synthetic receptor function. Forexample, synNotch molecules with an extracellular anti-mesothelin scFvdisplayed an improved threshold to activation when this extension wasincorporated (FIG. 88A).

The GFP and the CD19 detecting synNotches were tested against ligandpresented in different formats—soluble, cell-surface expressed, and ciscell surface expressed (i.e. on the same cell as the receptor) (FIG.86C). It was found that the synNotches transduce the signal only whentheir ligand is presented on an opposing surface; no activation wasdetected when the cognate ligand was in solution or presented on thesame surface as the receptor (cis). Interestingly, when the ligand iscis-presented on the cell that expresses the receptor, the synNotchreceptors display blunted activation (FIG. 86C, column “cell” vs“cell+cis”, and FIG. 88B-C) a feature known as cis-inhibition in nativeNotch.

On the intracellular side, it was shown that transcriptional repressionas well as activation can be directed. To this end, versions of thereceptors that have a transcriptional repressor domain (KRAB) fused to aGal4DNA-binding domain as intracellular domain were generated. The cellsthat express this synNotch respond by down-regulating the reporter genewhen co-cultivated with sender cells (FIG. 86B).

Other intracellular domains were also inserted into syn-Notch, includingCre recombinases, and master transcription factors such as MyoD andSnail. While some of these showed regulated activity, in general theseactivities were quite low, most likely because the synNotch receptoroutput domain functions stoichiometrically and does not showamplification. Thus this receptor system, may work better for highlyamplified outputs.

Synthetic Notch Receptors Work in Diverse Primary Cells: Neurons &Immune Cells.

SynNotch cell lines showed robust activation upon receptor activation.To test if this result is cell-line specific or not, synNotch receivercell lines were produced by engineering a series of established celllines: epithelial MDCK cells, L929 and C3H mouse fibroblasts cell lines,HEK293 human epithelial cell lines, Jurkat T cells. All of theminvariably showed a robust induction of reporter gene activation uponsynNotch receptor activation (FIG. 89B). Importantly, primary cells canalso be engineered with synNotch receptors, and in this way they becomeresponsive to novel ligand stimulation. In FIG. 89A (and FIG. 90) it isshown that primary hippocampal neurons that express the synNotch and thereporter can be induced to express a GFP reporter cassette by contactwith ligand-expressing sender cells.

Synthetic Notch Receptors can Regulate Diverse Cellular Behaviors in aSpatially Controlled Manner.

SynNotch receiver cells can detect whether the neighbor cells areexpressing the ligand or not. Whether synNotch receiver cells placed inan epithelial cell layer could be spatially induced to express areporter gene was tested. When ligand-expressing sender cells aresparsely plated in a layer of receiver cells, only the receiver cellsthat are directly contacting the sender cells activate the syntheticNotch receptor, as visible by the ring of blue cells (activatedreceivers) around the green sender cells, while the receiver cellsfurther away stay inactive (FIG. 91A).

Whether this synthetic cell-cell signaling could be used to regulatecell fate in a spatially controlled manner was then tested.Specifically, whether syn-Notch receptor could be used to induceexpression of a cell fate master regulator such as MyoD, a controller ofmuscle cell fate, was tested. As shown in FIG. 89B, transdifferentiationof synNotch fibroblasts can be induced only in the vicinity ofligand-expressing cells. In this experiment, CD19-expressing cells arelocally plated first, and then anti-CD19-synNotch fibroblasts areoverlaid uniformly. Where the synNotch receptor engages with its ligandthe receiver fibroblasts upregulate myoD, the prototypical mastertranscription factor for myogenesis, which leads to transdifferentiationand formation of myotubes only locally (green channel from myoD-GFP inreceiver cells spatially overlap with blue channel from BFP in sendercells, FIG. 89B; see FIG. 92A for a time-course).

In another example, that synNotch could be used to controlepithelial-to-mesenchymal transition was demonstrated. The anti-GFP (orthe anti-CD19) was used to control expression of the EMT masterregulator gene snail. In these epithelial cells, epithelial tomesenchymal transition was induced by exposing them to ligand-GFPexpressing cells, but not non-ligand expressing cells (FIGS. 91C and92B).

It was also shown that synNotch can be used to control spatialself-organization of multicellular assemblies. For these experiments,engineered fibroblasts were cultured as spheroids in low adhesiveplates, a setup where relative cell-cell adhesion strength dictates thegeometry. Receiver cells expressing anti-CD19 synNotch induce ofE-cadherin upon stimulation by sender cells expressing CD19. At thebeginning of the experiment, CD19-sender and anti-CD19-synNotch receivercells are randomly mixed (FIG. 91D, left). The induction of E-cadherinexpression downstream of the synNotch in the receiver cells increasestheir adhesion potential. The activated receiver cells now adherestrongly with one another and move in the inside of the spheroid,leaving the sender cells on the outer layer (FIG. 91D). As a control, itwas shown that this inside-out asymmetry of sender and receiver cellsself-organizes when only a fluorescent protein is activated in responseto synNotch activation, in place of E-cadherin (FIG. 92C).

Synthetic Notch Receptors are Orthogonal to Each Other and Native Notch.

Whether multiple synNotch receptors could function within the same cellwithout crosstalk was explored. Orthogonal function would allow the cellto elaborate different outputs according to the presence or absence ofmultiple inputs. It was hypothesized that synNotch receptors mightfunction orthogonally to one another because of the mechanism ofsignaling—there are no common intermediates (e.g. an activated kinase)that could yield cross talk, if the intracellular transcriptionalregulators in the different receptors are distinct. If there was anycross talk between the synNotch and the endogenous Notch signaling wasfirst tested. To show this, cells were engineered to report on theanti-CD19-synNotch as well as the full-length-Notch with differentfluorescent proteins. When the cells were stimulated with only Delta,only the full-length-Notch response was activated; the same applies tothe synNotch response to CD19: The two pathways show independentactivation (FIG. 93A).

Whether two different synNotch receptors could function independently inthe same cell was then tested. In FIG. 93B the results of stimulationwith single as well as combined ligands for two independent synNotchreceptors, an anti-CD19 receptor and an anti-GFP receptor, werereported. Each is linked to a different intracellular transcriptionactivation domain (Gal4 and tTA respectively), which in turn drives adistinct reporter fluorescent protein. When activated by CD19, only theanti-CD19-synNotch response is activated; conversely, when activated byGFP-expressing sender cells, only the anti-GFP-synNotch response isactivated. Importantly, when the cells are stimulated by both CD19 andGFP the two responses are activated together (FIG. 94B). Thus multiplesynNotch receptors can work in the same cell as insulated signalingtransduction pathways.

Multiple synNotch Receptors can be Used to Engineer Cells thatCombinatorially Integrate Multiple Inputs.

Multiple synNotch receptors can be used in the same cell to generatediverse responses. The engineering of cells that integrate combinatorialenvironmental cues and respond only when certain criteria are met weredesigned. In particular, the generation of cells that would respond onlyin the presence of two different antigens on their environment, butwould not respond to each one alone, were concentrated on. To achievethis, double synNotch expressing cells with anti-CD19 and anti-GFPextracellular domains: the activation of each synNotch activates a halfof a split-Gal4 protein, were engineered. It this way, when the twoinputs are presented to the receiver cell alone no activation is visible(FIG. 93C, columns 1-3). Only when the two receptors are activated isthe split molecule reconstituted and the response in receiver cellsinduced (FIG. 93C, last column).

Engineering Cascades of Cell-Cell Signaling with Multiple synNotchReceptors.

With multiple synNotch receptors for cell-cell communication,multicellular signaling systems of cell-cell communication weredesigned. In particular, the induction of self-organized multi-layerspatial patterning in epithelial cell layers was focused on. For this,the receiver cells have two synNotches, one of which induces the ligandfor the other. In this particular example, the anti-GFP-synNotch inducesCD19 ligand expression (and the reporter protein mCherry); and theanti-CD19-synNotch induces the reporter protein tagBFP. Then, to startthe induction of the cascade of layers, GFP sender cells are seededsparsely in a monolayer of the double synNotch receiver cells. Thiscircuit enabled the formation of two concentric rings of activationaround the sender cell islands: the first neighbors become red after theactivation of the anti-GFP-synNotch, and they become CD19 sender (FIG.94A, Day1). The cells one diameter further from the sender cells can nowrespond to the CD19 ligand expressed by the first neighbors, generatingthe double-ring pattern of FIG. 94A, Day2. In this way, two differentcell types are produced in a spatially controlled manner from a uniformpopulation of receiver cells (FIG. 94C). Thus multiple syn-Notchreceptors can be used to engineer more complex multistep patterningresponses, akin to what is observed in natural developmental processes(FIG. 94B).

FIG. 85. Modular Configuration of synNotch Receptors

(FIG. 85A) Conceptual design of synNotch receptor systems, as asuccessive engineering starting from wild-type Notch, to Notchreporters, to synNotch receptors. The latter exploit the flexibility ofintracellular orthogonal transcription factor of the notch reporter, andbuild a platform that responds to novel inputs by replacing theextracellular domain.

(FIG. 85B) Modularity of the platform: extracellular, transmembrane andintracellular domains abstracted from the Notch domain structure can beswapped with diverse domains on the outside (antibody based, or peptidetags are shown) and diverse effector in the inside (transcriptionalactivator with different DNA-binding domains are shown, as well as atranscriptional repressor).

FIG. 87 Provides Data Related to FIG. 85.

(FIG. 87A) Series of synNotch receiver cells with differentextracellular and intracellular domains show activation upon stimulationwith cognate ligand. Cells are mouse fibroblasts line L929, and arestimulated either by plate-bound stimulus or with sender cellsexpressing the ligand, as indicated in figure.

(FIG. 87B) Time course of activation of receiver cells upon sender cellsaddition (left); after full activation is reached, removal of sendercells induces inactivation of receiver cells (right).

(FIG. 87C) Dose response of activation of receiver cells with differentamount of plate-bound ligand. Myc-tag synNotch (left) and anti-GFPsynNotch (right) receiver cells are exposed to increasing amount ofligand; the reporter integrated intensity at 24h is shown in the graphs.

(FIG. 87D) SynNotch expression levels linearly influence the intensityof reporter activation. In the graph is shown reporter intensity againstreceptor expression levels for n=24 clonal population of fibroblastsexpressing the synNotch with myc-tag extracellular domain, inducing GFPas reporter. The blue dots are data from unstimulated cells, the greendots are data recorded after stimulation. A positive correlation betweenreceptor expression and GFP intensity is appreciable.

FIG. 86. SynNotch Receptors can be Used to Program Contact-DependentTranscriptional Regulation.

(FIG. 86A-B). Synthetic notch receptors can be used to detect endogenousdisease antigens and increase or decrease transcription of a reportergene.

(FIG. 86A) Mouse fibroblasts (L929 line) with anti-CD19/tTA synNotch arecultivated with sender cells expressing Delta, or CD19, or CD19 in thepresence of the gamma-secretase inhibitor DAPT. FACS plots of theresulting GFP intensity in receiver cells are shown.

(FIG. 86B) Mouse fibroblasts (L929 line) with anti-CD19 synNotch with atranscriptional repressor intracellular domain are co-cultivated with orwithout sender cells. The receiver cells express constitutively GFPdownstream of a SV40/UAS combined promoter. FACS plot of receiver cellsGFP intensity is shown, both in presence of sender cells with or withoutCD19 on the membrane.

(FIG. 86C) Mouse fibroblasts (L929) lines expressing synNotchstimulation with ligands in different formats. Left: anti-GFP/tTAsynNotch receiver cells are simulated with GFP either soluble, orsender-cell presented, or cis-presented on the receiver cell. The bargraph shows the reporter activation in the presence of the variousligand presentation choices: activation happens when the ligand ispresent on an opposing surface only. Right: myc-tagged anti-CD19/tTAsynNotch receiver cells are stimulated with ligands in differentformats, either anti-myc soluble antibody, or CD19 from sender cells orCD19 in the same receiver cell (cis). The bar graph shows the activationof the reporter: activation is induced when the ligand is presented onan opposing surface only.

FIG. 88. Related to FIG. 86.

(FIG. 88A) Addition of an EGF-repeat on the extracellular domain reducesbasal activation in the anti-mesothelin synNotch. Anti-mesothelinsynNotch receptors with or without an EGF repeat before theanti-mesothelin ScFv are introduced in mouse L929 fibroblasts. Theactivation of these receptors activates a GFP reporter. Data shown areFACS plots in two scenarios: up, without the EGF repeat, the inductionof the reporter is constitutive even in the absence of the ligand;bottom, with the EGF repeats, the basal reporter activation is abolished(OFF line), and the induction brings the GFP intensity to the ON state.

(FIG. 88B) Pulsed activation. To stimulate anti-GFP synNotch receivercells for a short amount of time, receiver cells are seeded on theplated for 24h and then incubated with suspension sender cells (K562sexpressing transmembrane GFP) for 1h or 4h; after that, suspensionsender cells washed away, and at t=24h from the first addition of sendercells the fluorescence in receiver cells is measured by FACS. Bar graphsare integrated fluorescence response of at least 10,000 cells for eachcondition. Data are average and standard error. Data are for anti-GFPLaG17 and LaG16/2 extracellular domains.

(FIG. 88C) FACS plots for the cis-inhibition results of main FIG. 86C.Plots are number of cells against reporter fluorescence of at least10,000 cells for each condition. Sender cells are engineered K562s andreceiver cells are engineered L929 mouse fibroblasts. On the left, datafor the anti-GFP synNotch are shown; on the right, data for theanti-CD19 synNotch.

FIG. 89. SynNotch Receptors Function in Diverse Cell Types, IncludingNeurons and Lymphocytes.

(FIG. 89A) Hippocampal neurons. Primary hippocampal neurons aredissociated from E18 rat embryos and are nucleofected to express ananti-CD19 synNotch and a coupled GFP reporter. Neurons are plated onglass-bottom 35 mm culture dish coated with Poly-D-Lysine and Laminin. 2hours after neuron plating, sender cells (K562s) are added to theculture to form co-culture system. Images are taken from live cells atday 4 after plating. On the right, representative images for neuronsthat are co-cultured with plain K562 cells (upper panel) or withCD19+K562 sender cells (Bottom panel) are shown. Neurons thatco-cultured with ligand presenting sender cells have significantlyhigher GFP expression than with control cells.

(FIG. 89B) T cell line. Jurkat T cell line is engineered to stablyexpress an anti-CD19/tTA synNotch receptor, that drives GFP as reporter.Data on the right show fluorescence of the Jurkat cells upon stimulationwith CD19+ or CD19− sender cells (K562s) at t=24h. T cells are activatedonly when they see the synNotch cognate ligand.

FIG. 90. Related to FIG. 89.

(FIG. 90A) Demonstration of the experiment and constructs that areexpressed in neurons. Primary hippocampal neurons are disassociated fromE18 rat embryos and are nucleofected with constructs that express thecNotch receptor as well as the TetO-GFP reporter. Neurons are plated onglass-bottom 35 mm culture dish coated with Poly-D-Lysine and Laminin. 2hours after neuron plating, K562 sender cells are added to the cultureto form co-culture system. Images are taken from live cells at day 4after plating.

(FIG. 90B) Distribution of the GFP fluorescent intensity in 100 neuronsfor each treatment. GFP intensity is calculated from the fluorescenceconfocal images.

(FIG. 90C) Quantification of the average GFP fluorescent intensity fromabout 100 neurons for each treatment.

FIG. 91. SynNotch Receptors Yield Spatial Control of Diverse CellularBehaviors

(FIG. 91A) Boundary detection in epithelial monolayer. Epithelial cells(MDCKs) are engineered as follows: sender cells express an extracellularGFP linked to a transmembrane domain; receiver cells express theanti-GFP synNotch with LaG17 anti-GFP nanobody as extracellular domain,and Gal4-VP64 as intracellular domain, alongside a UAS→BFP reporterconstruct. Sender and receiver in a 1:50 ratio are mixed together andconfocal images are taken at 48h after plating of confluent monolayer.Representative pictures of high magnification and low magnification areshown, alongside with a representative line of intensity of fluorescenceover distance. Only receiver cells that are in contact with the greensender cells turn on the blue reporter, forming a ring around the sendercells.

(FIG. 91B) synNotch activation of a myogenesis master regulator (myoD)in fibroblasts induce transdifferentiation in a spatially controlledmanner. C3H mouse fibroblasts are engineered as follows: sender cellsexpress extracellular CD19 linked to a transmembrane domain, plus atagBFP marker; receiver cells express the anti-CD19 synNotch with tTAintracellular domain, alongside a TRE→myoD reporter construct and aconstitutive mCherry marker. Sender fibroblasts are plated first in alimited region of the plate; after 1h, the sender cells are attached tothe plate, and the receiver cells are plated to cover all the glassplate. Images shown are at 48h after co-culture (see FIG. 92 for a timecourse). GFP channel shows the induction of myoD-GFP in a region thatoverlaps with the blue channel, marking the sender cells. An highermagnification of the field for the green channel is shown on the right.

(FIG. 91C) synNotch can induce epithelial to mesenchymal transition incultured epithelial cells. Epithelial cells (MDCKs) are engineered asfollows: receiver cells express the anti-GFP synNotch with LaG17anti-GFP nanobody as extracellular domain, and tTA as intracellulardomain, alongside a TRE→Snail-ires-BFP effector construct. Sender cellsare GFP-expressing K562s. Representative bright field microscope imagesof epithelial cells before and after the addition of sender cells areshown. See FIG. 92 for quantification.

(FIG. 91D) synNotch induction of adhesion in fibroblasts can governsymmetry-breaking rearrangements in fibroblasts spheroid cultures. L929mouse fibroblasts are engineered as follows: sender cells expressextracellular CD19 linked to a transmembrane domain, plus a tagBFPmarker; receiver cells express the anti-CD19 synNotch with tTAintracellular domain, alongside a TRE→E-cadherin-GFP effector construct,and a constitutive red marker (mCherry). Fluorescence signal ascollected with microscope is shown at t=0 (left) and at t=20h (right)for a representative spheroid. At t=0 cells are mixed; at t=20h receivercells induce E-cadherin (green channel) and sort in the inner layer ofthe spheroid (red), whereas the sender cells (blue) are on the outside.

FIG. 92. Related to FIG. 91

(FIG. 92A; Relative to FIG. 91B) C3H mouse fibroblasts are engineered asfollows: sender cells express extracellular CD19 linked to atransmembrane domain, plus a tagBFP marker; receiver cells express theanti-CD19 synNotch with tTA intracellular domain, alongside a TRE→myoDeffector construct and a constitutive mCherry marker. Sender fibroblastsare plated first in a limited region of the plate; after 1h, the sendercells are attached to the plate, and the receiver cells are plated tocover all the glass plate. Images are taken every 10h on the right.

Below, a representative field of an experiment of co-culture of senderand receiver cells (up) or receiver cells alone (bottom) is shown.

(FIG. 92B; Relative to FIG. 91C) Epithelial cells (MDCKs) are engineeredas follows: receiver cells express the anti-GFP synNotch with LaG17anti-GFP nanobody as extracellular domain, and tTA as intracellulardomain, alongside a TRE→Snail-ires-BFP effector construct. Sender cellsare GFP-expressing K562s. FACS plots of receiver cells BFP signal isshown in presence of no sender cells, of sender cells expressing anunrelated ligand, and of sender cells expressing the cognate ligand GFP.Quantification of the fluorescence is provided on the bar graph.

On the far right, the bar graph reports the E-cadherin expression levelsin the receiver cells or of parental cells in the various conditions.E-cadherin levels drops only in the presence of the cognate antigenGFP-expressing sender cells.

(FIG. 92C; Relative to FIG. 91D)

Upper panel: L929 mouse fibroblasts are engineered as follows: sendercells express extracellular CD19 linked to a transmembrane domain, plusa tagBFP marker; receiver cells express the anti-CD19 synNotch with tTAintracellular domain, alongside a TRE→E-cadherin-GFP effector construct,and a constitutive red marker (mCherry). Fluorescence signal ascollected with microscope is shown at t=20h for a representativespheroid with sender and receiver cells, and for one with receiver cellsonly as indicated. The green fluorescence is induced in the receivercells only in the presence of the sender cells (E-cad channel, farright).

Lower panel: L929 mouse fibroblasts are engineered as follows: sendercells express extracellular GFP linked to a transmembrane domain;receiver cells express the anti-GFP synNotch with tTA intracellulardomain, alongside a TRE→mCherry reporter construct, and a constitutiveblue marker (tagBFP). Fluorescence signal as collected with confocalmicroscope is shown at t=0 t=20h for a representative spheroid withsender+receiver cells, and for one with only receiver cells. The redfluorescence is induced in the receiver cells only in the presence ofthe sender cells (mCherry reporter channel, far right). Norearrangements of sender and receiver cells are appreciable.

FIG. 93. SynNotch Receptors are Orthogonal to One Another and can beUsed for Combinatorial Regulation

(FIG. 93A) synNotch and wild-type Notch activate orthogonal signalingpathways. L929 mouse fibroblasts receivers are engineered to express (i)the wild-type Notch receptor with a tTA intracellular domain and aTRE→GFP reporter, and (ii) a synNotch receptor with anti-CD19extracellular domain and Gal4-VP64 intracellular domain, and aUAS→tagBFP reporter. The graph on the right reports the receiver cellfluorescence signal for the BFP and the GFP reporters in differentconditions: black dots are untreated cells, blue dots receiver cellsstimulated with CD19 expressing senders, orange dots are receiver cellsstimulated with delta senders, and red dots are receiver cellsstimulated with sender cells expressing both CD19 and delta. Sendercells are mouse L929 fibroblasts.

(FIG. 93B) multiple synNotches are orthogonal one another. L929 mousefibroblasts receivers are engineered to express (i) the anti-CD19synNotch receptor with a tTA intracellular domain and a TRE→BFPreporter; and also (ii) the synNotch receptor with anti-CD19extracellular domain and Gal4-VP64 intracellular domain, and aUAS→mCherry reporter. The graph on the right reports the receiver cellfluorescence signal for the BFP and the GFP reporters in differentconditions: black dots are untreated cells, red dots are receiver cellsstimulated with CD19 expressing senders, green dots are receiver cellsstimulated with GFP senders, and blue dots are receiver cells stimulatedwith sender cells expressing both GFP and CD19. Sender cells are K562s.

(FIG. 93C) Cells engineered with two synNotches can respond only whenboth the inputs are present. L929 mouse fibroblasts receivers areengineered to express (i) the anti-CD19 synNotch receptor with a tTAintracellular domain and a TRE promoter that drives the expression ofthe DNA-binding domain (DBD) of Gal4 fused to a leucine zipper domain,and (ii) the synNotch receptor with anti-GFP extracellular domain andthe VP64 transcriptional activation domain fused to a complementaryleucine zipper as intracellular domain, and (iii) a Gal4-responsivepromoter driving a red fluorescent protein (mCherry). The graph on theright shows the normalized mCherry fluorescence collected from receivercells in co-culture with different sender cells (K562s), that expresseither the two ligands alone (GFP or CD19), or both ligands together.Activation occurs only in the presence of both the inputs.

FIG. 94. Multiple synNotch Receptors can be Used to GenerateMulti-Layered Self-Organizing Epithelial Patterns.

Epithelial cells (MDCKs) are engineered as follows: sender cells expressextracellular GFP linked to a transmembrane domain; receiver cellsexpress (i) the anti-GFP synNotch with tTA intracellular domain,alongside a TRE→CD19-mCherry effector cassette; (ii) the anti-CD19receptor with Gal4-VP64 intracellular domain, and a UAS→tagBFP reporter.

(FIG. 94A) Representative images are shown for the epithelial layer ofsender cells and receiver cells co-cultivated at a 1:50 ratio for 10h(START) 34h (DAY 1) and 58h (DAY2).

(FIG. 94B) Multiple images of different fields of view of the co-cultureat day 2.

(FIG. 94C) Representative quantification of the fluorescence signal ascalculated from the fluorescence images for a pattern around sendercells at day 2.

FIG. 95. Modularity of synNotch Receptors Expands Sensing/ResponseEngineering of Mammalian Cells.

(FIG. 95A) Putative structural mechanism of activation of the synNotchreceptors. The LNR domains mask the protease cleavage site in theunbound conformation (left). When the ligand engages the receptor, thesite is exposed and this event start the transduction (right).

(FIG. 95B) Alignment of LNR-containing molecules from early metazoan.

(FIG. 95C) The modularity of the synNotch receptor platform allow theuser to specify the extracellular cues the cells now respond to, as wellas the cellular responses that are induced downstream of receptoractivation.

Example 4: Engineering T Cells with Customized Therapeutic ResponsePrograms Using Synthetic Notch Receptors Material and Methods

The following materials and methods apply to the results described inExample 4 unless otherwise indicated.

synNotch Receptor and Response Element Construct Design

synNotch receptors were built by fusing the CD19 scFv, LaG17 (loweraffinity), or LaG16_2 (high affinity) GFP nanobody to the mouse Notch1(NM_008714) minimal regulatory region (Ile1427 to Arg1752) and Gal4VP64.All synNotch receptors contain an n-terminal CD8a signal peptide(MALPVTALLLPLALLLHAARP (SEQ ID NO:129)) for membrane targeting and amyc-tag (EQKLISEEDL (SEQ ID NO:75)) for easy determination of surfaceexpression with α-myc A647 (cell-signaling #2233). The receptors werecloned into a modified pHR′ SIN:CSW vector containing a PGK promoter forall primary T cell experiments. The pHR′ SIN:CSW vector was alsomodified to make the response element plasmids. Five copies of the Gal4DNA binding domain target sequence (GGAGCACTGTCCTCCGAACG (SEQ IDNO:130)) were cloned 5′ to a minimal CMV promoter. The human IL-2,IL-10, Tbet, or TRAIL codon optimized mRNA sequence was cloned into aMCS downstream of the Gal4 inducible promoter and 5′ of an IRES mCherryreporter. All constructs were cloned via In-Fusion cloning (Clontech#ST0345)).

Primary Human T cell Isolation and Culture

Primary CD4+ and CD8+ T cells were isolated from anonymous donor bloodafter apheresis by negative selection (STEMCELL Technologies #15062 &15063). Blood was obtained from Blood Centers of the Pacific (SanFrancisco, Calif.) as approved by the University Institutional ReviewBoard. T cells were cryopreserved in RPMI-1640 (UCSF cell culture core)with 20% human AB serum (Valley Biomedical Inc., #HP1022) and 10% DMSO.After thawing, T cells were cultured in human T cell medium consistingof X-VIVO 15 (Lonza #04-418Q), 5% Human AB serum and 10 mM neutralizedN-acetyl L-Cysteine (Sigma-Aldrich #A9165) supplemented with 30 units/mLIL-2 (NCI BRB Preclinical Repository) for all experiments of Example 4.

Lentiviral Transduction of Human T Cells

Pantropic VSV-G pseudotyped lentivirus was produced via transfection ofLenti-X 293T cells (Clonetech #11131D) with a pHR′ SIN:CSW transgeneexpression vector and the viral packaging plasmids pCMVdR8.91 and pMD2.Gusing Fugene HD (Promega #E2312). Primary T cells were thawed the sameday, and after 24 hours in culture, were stimulated with Dynabeads HumanT-Activator CD3/CD28 (Life Technologies #11131D) at a 1:3 cell:beadratio. At 48 hours, viral supernatant was harvested and the primary Tcells were exposed to the virus for 24 hours. At day 4 post T cellstimulation, Dynabeads were removed and the T cells expanded until day 9when they were rested and could be used in assays. T cells were sortedfor assays with a FACs ARIA II.

Cancer Cell Lines

The cancer cell lines used were K562 myelogenous leukemia cells (ATCC#CCL-243), Daudi B cell lymphoblasts (ATCC #CCL-213), and HCT115 coloncancer cells (ATCC #CCL-247). K562s were lentivirally transduced tostably express human CD19 at equivalent levels as Daudi tumors. CD19levels were determined by staining the cells with α-CD19 APC (Biolegend#302212). K562s were also transduced to stably express surface GFP (GFPfused to the PDGF transmembrane domain). All cell lines were sorted forexpression of the transgenes.

In Vitro Stimulation of synNotch T Cells

For all in vitro synNotch T cell stimulations, 2×10⁵ T cells wereco-cultured with sender cells at a 1:1 ratio. After mixing the T cellsand sender cells in round bottom 96-well tissue culture plates, thecells were centrifuged for 1 min at 400×g to force interaction of thecells and the cultures were analyzed at 24 hours for reporter expressionor expression of custom gene induction via flow cytometry with a BD LSRII. All flow cytometry analysis was performed in FlowJo software(TreeStar).

Luminex MAGPIX Cytokine Quantification

Primary CD4+ T cells expressing the α-CD19 synNotch Gal4VP64 receptorand 5×Gal4 response elements controlling either human IL-2 or IL-10expression were stimulated as described above with K562 myelogenousleukemia cells (CD19- or CD19+). As references, CD4+ T cells expressingthe α-CD19 4-1BBζ CAR were stimulated along with untransduced T cellsstimulated with α-CD3/CD28 Dynabeads at a 1:3 ratio. The supernatant wascollected at 24 hours and analyzed with a Luminex MAGPIX (Luminex Corp.)Human Cytokine Magentic 25-plex Panel (Invitrogen ref #LHC0009M)according to the manufacturer's protocol. All cytokine levels werecalculated based on standard curves with xPONENT software (LuminexCorp.).

IL-2 Intracellular Cytokine Staining and CD69 Staining

synNotch T cells controlling IL-2 production were assayed to determineif they basally produced IL-2 by intracellular cytokine stain (ICS). ThesynNotch T cells and untransduced T cell controls were cultured for 6hours in the presence of GolgiPlug (BD Biosciences #555029). The T cellswere then stained with a-IL-2 FITC (BD #340448) with a BD BiosciencesICS kit (#555028). The levels of IL-2 were analyzed via flow cytometrywith a BD LSRII.

To assess whether synNotch receptors activated the T cells, the T cellswere stained after stimulation for the activation marker CD69. CD69expression was determined by staining the cells with α-CD69 APC(Biolegend #310910).

synNotch Driven T Cell Differentiation

Primary human CD4+ T cells were stimulated with Dynabeads HumanT-Activator CD3/CD28 as described above. To differentiate T cells intothe T_(h1) subset during the activation, the cells were cultured asdescribed above but with the addition of 2.5 ng/mL recombinant IL-12(R&D Systems) and 12.5 μg/mL a-IL-4 clone MP4-25D2 (BD Pharmigen#554481). IL-12 and a-IL-4 were added at least twice weekly. Inparallel, primary CD4 T cells were lentivirally transduced to expresshuman Tbet T2A mCherry (TBX21, NCBI #EAW94804.1) and cultured normallyin T cell medium supplemented with IL-2. CD4+ T cells expressing theα-CD19 synNotch Gal4VP64 receptor and 5×Gal4 response elementscontrolling Tbet T2A GFP expression were cultured in the presence ofCD19- or CD19+K562 sender cells 24 hours after viral transduction. ThesynNotch T cells were cultured in the presence of K562s in T cell mediumsupplemented with IL-2. All T cells were cultured for 11 to 14 days andthen subject to intracellular cytokine staining (ICS) to determine thepercentage of T_(h1) T cells.

For ICS, the T cells were first treated with 50 ng/mL Phorbal myristateacetate and 1 μg/mL ionomycin (both from Sigma) for 6 hours in thepresence of GolgiPlug. The T cells were then stained with a-Tbet BV421(Biolegend #644816) and a-IFNγ APC (Biolegend #502512). The levels ofTbet and IFNγ were analyzed via flow cytometry with a BD LSRII.

Sensitivity of Cancer Cell Lines to Recombinant TRAIL and synNotchDriven TRAIL Production in Primary T Cells

HCT116 colon cancer cells and K562s were treated with recombinant TRAIL(from 1 to 200 ng/mL, 1:2 dilution series) for 24 hours. The cells werethen harvested and stained with the live/dead stain, SYTOX Blue (ThermoScientific #S34857) and the fraction of dead cells was determined byflow cytometry on a BD LSR II. The level of the death receptor 4 (DR4)expressed by K562s was assessed by staining with a-TRAIL R1 (DR4) APC(Biolegend #307208).

For synNotch driven TRAIL cytotoxicity assays, primary human CD4+ Tcells were transduced to express the a-GFP nanobody (LaG17) synNotchGal4VP64 receptor and 5×Gal4 response elements controlling theexpression of LZ-TRAIL or cell surface wild-type TRAIL. The synNotchTRAIL killer cells were co-cultured with surface GFP+ or GFP− K562s for24 hrs and death was determined by staining with SYTOX Blue. Surfacelevels of TRAIL was determined by staining T cells with a-TRAIL (CD253)APC (Biolegend #308210). Production and secretion of LZ-TRAIL wasdetermined by TRAIL ELISA (R&D systems #DTRL00).

Xenograft Tumor Model, Cell Isolation, and Flow Cytometry

Animal studies were conducted with the UCSF Preclinical TherapeuticsCore under a protocol approved by the UCSF Institutional Animal Care andUse Committee. NOD scid gamma (NSG) (female, 8˜12 weeks old, JacksonLaboratory #005557) mice were used for all in vivo mouse experiments.Primary CD4+ and CD8+ T cells expressing the α-CD19 synNotch Gal4VP64receptor and 5×Gal4 response elements controlling human IL-2 IRESmCherry were sorted and used in the experiments.

The mice were injected on day 0 with 5×10⁶ CD19+ and CD19− K562ssubcutaneously on the right flank and left flank of the mice,respectively. The tumors were allowed to establish for 4 days and Tcells were injected via the tail vein (i.v.) on day 4 or intratumoral onday 8. The T cells were suspended in PBS for all injections. CD4+ andCD8+ synNotch T cells were injected at a 1:1 ratio. For i.v. injections,6×10⁶ total T cells were injected, and for intratumoral injections,5×10⁵ total T cells were injected.

Tumors were harvested at day 10 into RPMI supplemented with 1% FBS (UCSFCell Culture Core). The tumors were then minced by razor blade anddigested for an hour in RPMI with 0.1 mg/mL DNase (Roche #10104159001)and 0.2 mg/mL collagenase P (Roche #11249002001) at 37° C. Afterincubation, the digested tumors were passed over a 75 μm cell strainerand the tumor cells were collected by centrifugation. The cells werethen treated with red blood cell lysis buffer (Biolegend #420301) andwashed with PBS. The tumors were then stained with a LIVE/DEAD Green(Thermo Scientific #34969) and α-CD4 A647 (BD 557707) and α-CD8 BV786(BD #583823) to analyze the tumor infiltrating T cells. Expression ofIL-2 IRES mCherry was assessed in the CD4+ and CD8+ T cell populationswith a BD LSR II.

Statistical Analysis

Statistical significance was determined by Student's t test (two-tailed)unless otherwise noted. All statistical analysis for Example 4 wasperformed with Prism 6 (Graphpad) and p values are reported(n.s.=p>0.05, *=p≤0.05, **=p≤0.01, ***=p≤0.001, ****=p≤0.0001). Allerror bars represent either S.E.M. or S.D.

Results

synNotch Receptors can Drive Antigen-Induced Transcription in CD4+ andCD8+ Human Primary T Lymphocytes

The Notch receptor has three critical components: 1) the ligand-bindingepidermal growth factor (EGF) repeats, 2) the core regulatory regionthat controls cleavage of the receptor during activation, and 3) theNotch intracellular domain (NICD) that is released and regulatestranscription. To build a synNotch receptor platform that allows forfully customizable receptor targeting and transcriptional regulation,the Notch core regulatory region that controls ligand-dependent cleavageand activation was utilized as a minimal scaffold, but then appendedwith customized input recognition and output transcriptional modules(see Example 3). The Notch core regulatory region includes theLin12-Notch repeats (LNRs) that control the accessibility of the S2cleavage site to metalloproteases, the heterodimerization domains (HD),and transmembrane domain (TMD) that contains the y-secretase cleavagesite required for release of the Notch intracellular domain (NICD). Theextracellular EGF repeats, normally involved in recognition of thenatural ligand delta, were removed and replaced with a single-chainvariable fragment (scFv) directed towards the cancer antigen CD19 ornanobodies to orthogonal antigens, such as surface displayed GFP (FIG.96B-96C). The NICD that is normally required for transcriptionalregulation was replaced with the Gal4 DNA binding domain fused to thetetrameric viral transcriptional activator domain, VP64 (FIG. 96B-96C).This general approach can be used to engineer synNotch receptors to anysurface antigen of interest and link receptor activity to a customizedcellular output controlled by orthogonal transcription factors and theirassociated response elements (see Example 3). A range of otherextracellular and intracellular domains were also shown to function withsynNotch.

To show that synNotch receptors can function in relevant cell types forcell-based therapy, primary human CD4+ and CD8+ T cells were engineeredwith synNotch receptors directed towards the cancer-related antigensCD19 or to an orthogonal antigen—surface displayed GFP. CD4+ and CD8+ Tcells were engineered to express each synNotch receptor and theassociated promoter (5×Gal4 response elements) controlling expression ofa BFP reporter gene (FIG. 97). CD4+ and CD8+ T cells engineered with theα-CD19 synNotch receptor drove BFP reporter expression in 80 to 90% ofthe T cells within 24 hours of co-culture with cells expressing thecognate ligand CD19—either Daudi B cell lymphoblast tumors, whichnaturally express CD19, or K562 myelogenous leukemia cells withectopically expressed CD19 (FIG. 97A-97C and FIG. 103A-103C). These Tcells did not show BFP expression when unstimulated or treated withcells that did not express the cognate CD19 antigen. These data showthat synNotch receptors can function in a controlled andantigen-dependent manner in primary T cells, and can detect naturallevels of antigen on the surface of cancer cells. The synNotch receptorsalso have equivalent function in CD4+ and CD8+ T cells, which are oftenused in concert for T cell immunotherapies (FIG. 97C and FIGS. 103A and103B).

Whether T cells could be engineered to recognize orthogonal surfaceproteins was also tested by constructing synNotch receptors thatrecognized surface expressed GFP. Two a-GFP nanobodies that have low(Kd=50 nM) or high affinity (Kd=0.036 nM) (Fridy et al., 2014) wereused. These receptors stimulated reporter expression upon exposure toK562 cells expressing surface GFP (but not with cells lacking theantigen). The resulting transcriptional response was similar to thatobserved for the α-CD19 synNotch, highlighting the modularity of thesynNotch platform (FIG. 97D-97F and FIG. 103G-103I). The higher affinitya-GFP nanobody synNotch receptor drove a greater fraction of T cells toupregulate reporter expression upon exposure to K562 sender cells, whencompared to the lower affinity receptor, yet both receptors activatedgene expression in over half of the T cells (FIG. 97D-97F and FIG.103H). This suggests that varying the affinity of the synNotchligand-binding domain can fine tune the magnitude of the cellularresponse.

synNotch Receptors can Drive Customized T Cell Cytokine Profiles

Immune cells and tissues throughout the body secrete soluble proteinsknown as cytokines to communicate and regulate cell behavior and shapethe overall immune response. The specific cytokine profile is criticalfor eradicating pathogens and tumors. In many cases the different setsof cytokines have the opposite effect of suppressing the immuneresponse. Moreover, in certain scenarios where cytokines could boostimmunity towards cancers or suppress damaging inflammation in anautoimmune setting, these cytokines are absent. Precise control of whatcytokines therapeutic T cells secrete is desirable. However, in someinstances, when T cells are activated through CARs or the natural T cellreceptor, there is little control over the cytokines that are producedand often cytokine profile depends on the disease and activation contextas well as the receptor characteristics (FIG. 98A-98C). For many T celltherapies, it may be beneficial to bias towards the production ofspecific cytokines to tailor the immune response for a specific diseaseor therapeutic need.

With this in mind, CD4+ T cells were engineered with the α-CD19 synNotchreceptor and the corresponding transcriptional response elementcontrolling the expression of a single cytokine. Under these conditions,T cells selectively produced only a defined “a la carte” cytokineprofile in response to the CD19 antigen (FIG. 98D-98F). synNotchreceptors drove high-level production of the T cell stimulatory cytokineIL-2 with no basal secretion prior to antigen sensing (FIG. 104A-104D).The amount of IL-2 produced by synNotch activation is similar to what isproduced in response to CAR or TCR stimulation (α-CD3/α-CD28 beads) of Tcells (FIG. 104D). Unlike normal stimulation of T cells through the TCRpathway, synNotch-driven cytokine production does not lead to T cellactivation, shown by the lack of upregulation of the activation markerCD69 (FIG. 104E).

T cells were engineered to produce the immunosuppressive cytokine IL-10(FIG. 98D-98F and FIG. 104F-104H), which unlike IL-2, was absent fromthe cytokine profile of activated CAR T cells (FIG. 98C). Thishighlights an application of synNotch receptors—the ability to add ormodulate levels of a particular cytokine, even one that is not naturallyexpressed (FIG. 98 and FIG. 104). Moreover, such responses do notrequire normal activation of the T cell. The customizability andprecision of synNotch circuits in T cells should allow for local controlof immune system functions that rely on autocrine and paracrine cellularcommunication to effectively coordinate a response to disease.

synNotch Receptors can Drive Antigen-Dependent Skewing of T CellDifferentiation to the Anti-Tumor T_(h1) Fate

Another way to precisely shape the output of a therapeutic T cell is tocontrol its differentiation. Beyond producing protein effectors likecytokines, T cells undergo specific differentiation programs importantfor mounting an effective subtype immune response. These subtypedifferentiation programs are normally determined by the combination of Tcell activation, particular cytokines, and ultimately the regulation ofmaster regulator transcription factors that initiate the specific T cellfate. T helper cell 1 (T_(h1)) or T helper cell 2 (T_(h2)) T cells aretwo canonical CD4+ T cell fate choices that are controlled by the masterregulator transcription factors, Tbet and GATA3, respectively (FIG.99A). T_(h1) cells are important for cellular immunity towards pathogensand cancer whereas T_(h2) cells are involved in stimulation of antibodyproduction. In many diseases, the local environment skews thedifferentiation of T cells along the wrong path such that they arerendered ineffective. This is especially true in cancer where T cellscan be pushed into a suppressive phenotype, hampering the immuneresponse and leading to tumor expansion.

Given the importance of T cell fate choice for cancer clearance, ifsynNotch receptors could skew T cells to differentiate into IFNγproducing T_(h1) cells, important for anti-cancer immunity, wasinvestigated. IFNγ is critical for activation of innate immune cellsthat aid in tumor clearance, such as macrophages and dendritic cells,and direct exposure of cancer cells to IFNγ can enhance theirsusceptibility to the cytotoxic T cells. To skew T cell differentiation,CD4+ T cells were engineered with the α-CD19 synNotch receptor thatcontrolled the expression of the T_(h1) transcription factor, Tbet (FIG.99B). Since ectopic expression of Tbet is known to be sufficient todrive the T_(h1) fate choice in CD4+ T cells, it was reasoned thatsynNotch could provide antigen-dependent control over T_(h1) fateregulation by regulating the levels of Tbet in response to the tumorantigen CD19.

To test this, the engineered primary CD4+ T cells were co-cultured witheither CD19+ target K562 cells or CD19− control K562 cells for 11 daysto induce differentiation. As comparative controls, a matched populationof CD4+ T cells were either treated with a cocktail of T_(h1)differentiation agents (IL-12 and a-IL-4) or subject to Tbetconstitutive overexpression. These two conditions allowed for thecomparison of synNotch driven T_(h1) differentiation to previous goldstandards in the field. The engineered synNotch CD4+ T cells expressedTbet in response to CD19 within 24 hours of stimulation (FIG. 99C andFIG. 105A). After 11 days of long-term co-culture with CD19+K562s, the Tcells were stimulated with Phorbal myristate acetate (PMA) and ionomycinfor intracellular cytokine staining to reveal whether the T cells hadbecome T_(h1) cells. For the T cells that had been stimulated withCD19+K562 cell for 11 days, >60% were found to be IFNy+T_(h1) cells(FIG. 99D). This magnitude of skewed differentiation was equivalent towhat was observed with treatment with the T_(h1) differentiationcocktail and only slightly less than with constitutive Tbetoverexpression (FIG. 99E and FIG. 105A-105E). Thus, synNotch receptorscan be used to skew T cells to the anti-tumor T_(h1) fate, and could inprinciple be used to skew T cells to many of the known T cell fates(e.g. T_(h2), T_(reg), T_(hl7)) as long as the expression of thedefining master regulator transcription factor is sufficient for fatedetermination.

SynNotch Driven T Cell Delivery of Custom Therapeutics—TRAIL Production

Another important component of future T cell therapeutics is to engineerT cells with new capabilities that allow them to deliver customizedtherapeutic payloads, even ones that are non-native. Natural T cells orCAR T cells directly recognize infected cells or cancer cells and killthem through the delivery of lytic granules (FIG. 100A). However,natural T cell responses are often insufficient or too extreme toeradicate disease safely. Custom delivery of therapeutic payloads by Tcells, such as secreted biologics could aid in difficult to treatdiseases by, for example, locally enhancing cytotoxic activity or bypriming the site of disease to be recognized and killed by the immunesystem.

As a proof of principle experiment, the engineering of CD4+ T cells—a Tcell subset that is minimally cytotoxic—into a synthetic “killer T cell”by designing it to produce an apoptosis inducing payload wasinvestigated. The a-GFP nanobody synNotch receptor and response elementscontrolling production of Tumor Necrosis Factor Receptor ligand (TRAIL),an inducer of apoptosis and a cancer therapeutic, were used (FIG. 100B).T cells do not normally produce TRAIL upon TCR stimulation, therefore,if synthetically expressed in a controlled manner, this could aid intheir cytotoxic activity (FIG. 106A). Soluble forms of TRAIL areeffective at killing the highly susceptible colon cancer cell lineHCT116, but for other cancer lines like K562 cells, soluble TRAIL doesnot induce apoptosis even at high doses (FIG. 106A-106D). However, arecent study showed that if TRAIL is delivered in a membrane anchoredform (e.g., a supported lipid bilayer or liposome), it is more effectiveat inducing apoptosis, even for resistant cancer cells such as K562cells. Therefore, the CD4+ T cells were engineered to produce one of twoTRAIL variants: 1) a secreted form of TRAIL fused to the GCN4 trimericleucine zipper (LZ-TRAIL) known to be more potent than soluble monomericTRAIL, or 2) a natural surface displayed TRAIL (FIG. 106E 106H).

synNotch T cells driving TRAIL production were co-cultured withTRAIL-resistant K562 cells to determine if T cells were an effectivedelivery platform that enhanced the apoptotic effects of TRAIL. synNotchT cells drove cell surface TRAIL expression (FIG. 100C) and LZ-TRAILsecretion within 24 hours of co-culture (FIG. 106F-106H), but only cellsurface TRAIL initiated K562 cell death, indicated by their uptake ofthe live/dead stain SYTOX blue. In contrast, synNotch T cells thatsecreted LZ-TRAIL were not effective at killing the resistant K562cells, consistent with recent studies (FIG. 100D-100E).

Overall, these findings suggest that synNotch T cells can be efficientand effective delivery agents for therapeutics such as TRAIL. Anybiologic agent that has been ineffective or toxic when systemicallydelivered, might be more effective and safer if locally delivered inthis manner. synNotch engineered T cells have the potential to locallydeliver any genetically encoded therapeutic agents for enhancedeffectiveness and reduced systemic OFF-target toxicity.

In Vivo Expression of Cytokine in Solid Tumor Via synNotch ReceptorEngineered T Cells

Since synNotch receptors can precisely regulate a spectrum of T cellresponses in vitro, whether the receptors could selectively targetprimary human T cells in vivo to solid tumors and induce the delivery ofa custom payload such as the cytokine IL-2 was investigated. For theseexperiments, a bilateral K562 xenograft solid tumor model wasestablished in immunocompromised NOD scid gamma (NSG) mice where anon-target CD19− tumor and a target CD19+ tumor were implantedsubcutaneously in the left flank and right flank, respectively (FIG.101A). The tumors were allowed to establish for four days and then CD4+and CD8+ T cells engineered with the α-CD19 synNotch receptor andresponse elements in control of IL-2 expression and an IRES mCherryreporter were intravenously (i.v.) injected (FIG. 107A). After six daysthe tumors were harvested and the tumor infiltrating T cells wereanalyzed for expression of the IL-2 IRES mCherry reporter (FIG.101A-101C). Only the tumor localized T cells expressed the mCherryreporter for IL-2 expression in the target CD19 tumor, and the reporterlevel was similar to what was observed for the same T cells whenstimulated in vitro (FIG. 101A-101C and FIGS. 107B and 107C). While thefrequency of T cells was not high in the tumor (1 in 1000 cells for CD4+and 1 in 500 for CD8+ T cells), the activity of the T cells was highlyspecific to the target tumor (FIGS. 101B and 101C and FIG. 107D). Inaddition to i.v. injection of synNotch→IL-2 T cells, the T cells werealso directly injected into non-target and target tumors. The tumorswere then harvested and analyzed via flow cytometry two days afterinjection and also showed selective expression of the IL-2 reporter inthe target tumor at similar levels to matched in vitro stimulated Tcells (FIG. 107E). While the ability of synNotch T cells to infiltratethese tumors could still be improved, these data clearly show thatsynNotch receptors can target T cells to primary tumors and selectivelyinduce production of a therapeutic agent in a local manner. ThussynNotch engineered T cells could prove effective for delivery of awide-range of genetically encodable therapeutics that could benefit fromlocal delivery both to enhance effectiveness and reduce toxicity ofsystemic administration.

FIG. 96. The Potential to Engineer Customized Therapeutic T CellResponses Using synNotch Receptors.

(A) TCRs and CARs activate kinase-based signaling cascades that drivethe native T cell activation program providing little control overreshaping the T cell response. synNotch receptors recognize cell-surfaceantigens (e.g. disease related antigens) and directly regulate customtranscriptional programs with more precise control over the T cellresponse. Thus, synNotch receptors could be used to engineer a la carteresponses. (B) synNotch receptors may have a custom ligand bindingdomain (e.g. scFv or nanobody) that detects a cell-surface antigen ofinterest, the core regulatory region of Notch, and cytoplasmic domaincontaining an orthogonal transcription factor (e.g. Gal4 VP64). Thecorresponding response elements for the orthogonal transcription factorcontrolling custom transcriptional program may be engineered into the Tcell along with the receptor. (C) synNotch receptors with scFvs directedtowards the cancer-related antigens CD19 and nanobodies to theorthogonal antigen GFP were engineered and demonstrate the versatilityof the synNotch receptor platform.

FIG. 97. synNotch Receptors can Drive Antigen-Induced Transcription inCD4+ and CD8+ Human Primary T Lymphocytes.

(A) CD4+ AND CD8+ primary human T cells were engineered with the α-CD19synNotch Gal4VP64 receptor and 5×Gal4 response elements controlling theexpression of a BFP reporter. (B) Histograms showing selective inductionof the BFP reporter in α-CD19 synNotch receptor receiver T cells inresponse to stimulation with sender cells with CD19− or CD19+K562s orDaudi cancer cells (CD19+) after 24 hours of co-culture (representativeof ≥3 experiments). (C) Percentages of α-CD19 synNotch T cells thatupregulate the BFP reporter after 24 hours of stimulation with sendercells calculated from replicate data shown in panel B (n≥3 for allconditions, error bars=SEM). (D) CD4+ AND CD8+ primary human T cellswere engineered with a-GFP synNotch Gal4VP64 receptor affinity variantsand BFP reporter as in panel A. (E) Histograms showing selectiveinduction of the BFP reporter in a-GFP synNotch receptor receiver Tcells in response to surface GFP- or GFP+K562 sender cells after 24hours of co-culture (representative of ≥3 experiments, error bars=SEM).(F) Percentages of a-GFP synNotch T cells that upregulate the BFPreporter after 24 hours of stimulation with sender cells calculated fromreplicate data shown in panels H (n≥3 for all conditions, errorbars=SEM).

FIG. 103. Supplemental Data Related to FIGS. 97A-97F.

(A) Two dimensional dot plots of CD4+(left plot) and CD8+(right plot)primary human T cells transduced with the α-CD19 synNotch Gal4VP64receptor (myc tagged) and 5xGal4 response elements controllingexpression of BFP. The response element vector also contains a PGKpromoter that drives constitutive expression of mCherry to identify theT cells with the inserted response elements. All BFP reporter expressionanalysis was performed on T cells that had both the synNotch receptorand the corresponding 5xGal4 response elements controlling BFPexpression (population outlined in upper right box). (B) Histogramsshowing selective induction of the BFP reporter in synNotch receptorreceiver T cells in response to CD19+K562s or Daudi Tumor cells comparedto CD19− K562 control cells after 24 hours of co-culture (representativeof ≥3 experiments). (C) Histograms showing CD19 levels on K562s andDaudi tumor cells. Daudi tumors naturally express CD19 and K562sectopically express CD19 at similar levels. (D) Two dimensional dotplots similar to FIG. 103A for a-GFP nanobody synNotch Gal4VP64 receptorexpressing CD4 (top row) and CD8 (bottom row) primary human T cells.Each column is for a particular a-GFP nanobody affinity variant (LaG17,LaG16-2). All BFP reporter expression analysis was performed on T cellsin the upper right outlined gate as in FIG. 103A. (E) Histograms showingselective induction of the BFP reporter in synNotch receptor receiver Tcells in response to surface GFP+K562 cancer cells compared to surfaceGFP− K562s after 24 hours of co-culture (representative of ≥3experiments). (F) Histograms showing total GFP level in K562 cancercells expressing surface GFP compared to K562 GFP-controls.

FIG. 98. synNotch Receptors can Drive Antigen-Induced Custom CytokinePrograms.

(A) CAR activation drives T cells to produce a diverse set of cytokines.(B) A scatter plot showing the level (pg/mL) of 25 cytokines (see FIG.98C for list of cytokines) produced by primary human α-CD19 CAR CD4+ Tcells activated with target CD19+K562 cells (y-axis) or negative controlCD19− K562s (x-axis) after 24 hours of stimulation (n=3, errorbars=SEM). (C) The level of 25 cytokines produced by CD4+α-CD19 CAR Tcells stimulated by target CD19+K562s (n=3, error bars=SEM). (D) CD4+ Tcells were engineered with the α-CD19 synNotch Gal4VP64 receptor and thecorresponding response elements controlling the expression of eitherIL-2 or IL-10. The cells were co-cultured with target CD19+K562s orCD19− non-target K562s. (E) Scatterplots as in panel B showing theproduction of a single cytokine in response to CD19+K562 stimulation(n=3, error bars=SEM). (F) The level of cytokines produced by α-CD19synNotch T cells driving IL-2 or IL-10 production in response toCD19+K562 cells. Only the single cytokine (IL-2 or IL-10) is producedabove background levels (n=3, error bars=SEM).

FIG. 104. Supplemental Data Related to FIGS. 98A-98F.

(A) CD4+ human primary T cells were engineered with the α-CD19 synNotchGal4VP64 receptor and the associated 5×Gal4 response elements in controlof IL-2 production. (B) Two dimensional dot plot (left panel) of CD4primary human T cells transduced with the α-CD19 synNotch Gal4VP64receptor and 5×Gal4 response elements controlling expression IL-2expression IRES mCherry. The response element vector also contains a PGKpromoter that drives constitutive expression of BFP to identify the Tcells with the inserted response elements. T cells that had both thesynNotch receptor and the corresponding 5×Gal4 response elementscontrolling IL-2 IRES mCherry expression were sorted and used for allcorresponding assays in FIGS. 98 and 104. (upper right box). The leftpanel shows no basal induction of the IL-2 IRES mCherry reporter in dualpositive T cells compared to untransduced T cells. (C) Dot plots ofintracellular cytokine stains for IL-2 are shown for unstimulated CD4+and CD8+ T cells, unstimulated α-CD19 synNotch Gal4VP64 T cellscontrolling IL-2 production, and positive control T cells stimulatedwith PMA/ionomycin for 6 hours. (D) The basal and stimulated IL-2 levelsare given for supernatants harvested from untransduced CD4+ T cells,α-CD19 4-1BBζ CAR T cells, and α-CD19 synNotch Gal4VP64 T cellscontrolling IL-2 production (n=4). (E) CD69 levels (left column) andIL-2 IRES mCherry reporter levels in control CD4+ T cells stimulatedwith α-CD3/CD28 dynabeads and α-CD19 synNotch Gal4VP64 T cellscontrolling IL-2 production stimulated with CD19− or CD19+K562s. CD69 isnot upregulated on synNotch T cells upon stimulation with cognateantigen. (F) CD4+ human primary T cells were engineered with the α-CD19synNotch Gal4VP64 receptor and the associated 5×Gal4 response elementsin control of IL-10 production. (G) Equivalent data to panel B for CD4+primary human T cells transduced with the α-CD19 synNotch Gal4VP64receptor and 5×Gal4 response elements controlling expression of IL-10IRES mCherry expression. (H) Equivalent data to panel D for CD4+ primaryhuman T cells transduced with the α-CD19 synNotch Gal4VP64 receptor and5×Gal4 response elements controlling expression IL-10 IRES mCherryexpression. (I) Equivalent data to FIG. 104E for CD4 primary human Tcells transduced with the α-CD19 synNotch Gal4VP64 receptor and 5×Gal4response elements controlling expression IL-10 IRES mCherry expression.

FIG. 99. SynNotch Receptors can Drive Antigen-Dependent Skewing of TCell Differentiation to the Anti-Tumor T_(h1) Fate.

(A) Natural T cell Differentiation: When CD4+ T cells are activatedthrough engagement of pathogen-derived peptides presented by MHCmolecules on antigen-presenting cells they differentiate into particularT cell subtypes depending on the infection. T_(h1) and T_(h2) arecanonical CD4+ T cell fates that drive different immune responses.T_(h1) cells express the transcription factor Tbet, produce IFNγ, andaid in cellular immunity and tumor clearance. T_(h2)cells produce IL-4,an important cytokine for stimulation of antibody production by B cells.(B) SynNotch Driven T cell Differentiation: CD4+α-CD19 synNotch T cellswere engineered to regulate the expression Tbet and thus T_(h1) fatechoice by T cells. The synNotch T cells were co-cultured with targetCD19+ or non-target CD19− K562 cells for 11 days to determine ifsynNotch driven Tbet expression could skew CD4+ T cells to T_(h1) fatein a CD19-dependent manner. (C) Histograms showing the selectiveexpression of Tbet T2A EGFP after 24 hours of CD4+α-CD19 synNotch Tcells with CD19+K562s (representative of at least 3 experiments). (D)Two dimensional dot plots of intracellular stained CD4+α-CD19 synNotchGal4VP64 T cells for Tbet and IFNγ after 11 days of culture with eitherCD19+ or CD19− K562s. T cells were stimulated with PMA/Ionomycin for 4hrs prior to staining to drive cytokine production (representative of atleast 3 experiments). (E) The percentage of IFNγ+(T_(h1)) T cells after11 days of the indicated treatment (n≥3 for all treatments, errorbars=SEM, significance determined by student's t test, n.s. p>0.05).

FIG. 105. Supplemental Data Related to FIGS. 99A-99E.

(A) Quantification of replicate data from FIG. 99C. CD4+ T cells withα-CD19 synNotch Gal4VP64 receptor response elements controlling Tbet T2AGFP expression were co-cultured with CD19+ or CD19− K562s for 24 hrs.The percentage of T cells with Tbet T2A GFP expression is quantifiedshowing Tbet was only upregulated when the T cells were exposed toCD19+K562s (n=3). (B) Representative dot plot of CD4 T cells stimulatedwith α-CD3/CD28 dynabeads and intracellularly stained for the T_(h1)cytokine IFNγ after 11 days in culture. The IFNγ positive gate is boxedin red. (C) Representative dot plot similar to panel B for CD4+ T cellscultured for 11 days in T_(h1) differentiation conditions (IL-12+a-IL-4)(n=4). (D) Representative histograms of CD4 T cells with constitutiveoverexpression of Tbet T2A mCherry at 48 hours post transduction. (E)Representative dot plot of intracellular stains of CD4+ T cells withconstitutive overexpression of Tbet T2A mCherry for Tbet and IFNγ after11 days of culture (n=6). Dual positive Tbet and IFNγ T cells are boxedin red. (F) Representative microscopy of CD4+ T cells with the α-CD19synNotch Gal4VP64 receptor response elements controlling Tbet T2A GFPexpression (constitutively express mCherry) co-cultured with controlCD19− K562s. The synNotch T cells (red cells) do not associate with thecancer cells or upregulate the reporter of Tbet expression (GFP). (G)Representative microscopy as in panel F but the T cells were co-culturedwith CD19+ T cells. The synNotch T cells (red cells) form assemblieswith CD19+K562s and upregulate Tbet T2A GFP expression. The activatedsynNotch T cells are yellow due mCherry and GFP coexpression.

FIG. 100. SynNotch Driven TRAIL Production—Custom T Cell Delivery ofNon-Native Therapeutic.

(A) Natural T cell cytotoxicity: CD8+ cytotoxic T cells recognizeinfected cells via their TCR and directly kill the infected cell bycreating pores in the cell with perforin allowing for the delivery ofgranzymes that initiate programmed cell death. (B) SynNotch CustomizedKiller T cell: CD4+ T cells were engineered with the a-GFP synNotch thatcontrols the expression of the apoptotic regulator TRAIL in response tosurface GFP. (C) Histograms showing the selective expression of surfaceTRAIL after 24 hours of CD4+a-GFP synNotch T cells with surfaceGFP+K562s. (D) Histograms showing surface GFP+K562 cell death via uptakeof the dead stain SYTOX blue after 24 hr. co-culture with the indicatedT cell type (T cell:Target Cell Ratio=1:1). (E) Percentage target cellsurvival calculated from replicate data shown in panel D (n=4, errorbars=SEM).

FIG. 106. Supplemental Data Related to FIGS. 100A-100E.

(A) Rested CD4+ and CD8+ T cells were restimulated for 24 hrs withα-CD3/CD28 dynabeads and stained for cell surface TRAIL. Neither T cellsubset acutely upregulates TRAIL expression. (B) Cancer cells vary intheir sensitivity to recombinant TRAIL-mediated apoptosis. HCT116 coloncancers have the death receptors bound by TRAIL and are sensitive to lowlevel TRAIL treatment. K562s express the death receptors for TRAIL butare insensitive to TRAIL treatment. (C) Surface GFP− or GFP+ K562s orHCT116 cancer cells were treated with 0 to 200 ng/mL of recombinantTRAIL for 24 hrs and death was monitored via flow cytometry by stainingwith the dead stain SYTOX blue (n=4). (D) Histogram of K562s stained forthe death receptor 4 (DR4) bound by TRAIL. K562s express the receptortheir resistance to TRAIL-mediated apoptosis. (E) Representative dotplots of CD4+ T cells transduced with the LaG17 a-GFP nanobody synNotchGal4VP64 and 5×Gal4 response elements controlling expression of leucinezipper TRAIL (LZ-TRAIL left panel) or full-length surface TRAIL (rightpanel). The level of the mCherry reporter of TRAIL expression is shownbelow for the dual positive T cells and control untransduced T cells.(F) CD4+ T cells transduced with the LaG17 a-GFP nanobody synNotchGal4VP64 and 5×Gal4 response elements controlling expression LZ-TRAILwere co-cultured with surface GFP− or GFP+K562s for 24 hrs to determineif LZ-TRAIL is secreted only in response to GFP+K562s. (G) Histogramsshowing the level of mCherry reporter levels of TRAIL production insorted synNotch CD4 LZ-TRAIL T cells shown in FIG. 106E were co-culturedwith surface GFP= or GFP+K562s. The reporter was exclusively activatedin response to surface GFP+K562s. (H) TRAIL ELISA of supernatant fromsorted synNotch CD4 LZ-TRAIL T cells co-cultured with either surfaceGFP− or GFP+K562s for 24 hrs (n=2).

FIG. 101. In Vivo Local Expression of Cytokines at Solid Tumors ViasynNotch Receptor Engineered T Cell.

(A) NSG mice were subcutaneously injected with CD19− non-target K562sand target CD19+ in the left and right flank, respectively. α-CD19synNotch T cells in control of IL-2 iRES mCherry expression wereinjected into the mice after tumors were established and tumors wereharvested at the indicated timepoint to determine whether the synNotch Tcells had infiltrated the tumor and expression of IL-2 and mCherryreporter was induced. (B) Histograms of IL-2 IRES mCherry reporterlevels in tumor infiltrated CD4+ and CD8+ synNotch T cells injectedi.v., showing selective expression of the mCherry reporter in targetCD19+ tumors (data representative of 3 replicate mice). (C)Quantification of the percentage of tumor infiltrated T cells thatinduced expression of the mCherry reporter of IL-2 expression fromreplicate data shown in panel B (n=3, significance determined bystudent's t test ** p<0.01).

FIG. 107. Supplemental Data Related to FIGS. 101A-101C.

(A) Two dimensional dot plots of CD4+(left plot) and CD8+(right plot)primary human T cells transduced with the α-CD19 synNotch Gal4VP64receptor (myc tagged) and 5×Gal4 response elements controllingexpression of IL-2 IRES mCherry. The response element vector alsocontains a PGK promoter that drives constitutive expression of BFP toidentify the T cells with the inserted response elements. T cells in theupper right shaded box were sorted and used for all in vivo and in vitroexperiments. (B) Representative histograms of CD4+ and CD8+ synNotch Tcells in control of IL-2 expression stimulated in vitro with CD19− orCD19+K562s. IL-2 IRES mCherry expression occurred only in the presenceof CD19. (C) Quantification of the percentage CD4+ and CD8+ T cells thatinduced expression of the mCherry reporter of IL-2 expression fromreplicate data shown in FIG. 107B (n=3, significance determined bystudent's t test *** p<0.001 and **** p<0.0001). (D) Histograms of IL-2IRES mCherry reporter levels in tumor infiltrated CD4+ and CD8+ synNotchT cells injected intratumorally, showing selective expression of themCherry reporter in target CD19+ tumors (data representative of 3replicate mice). Protocol for intratumoral injection experiments isshown. Results are similar to what is observed for i.v. injected T cellsand in vitro stimulated T cells. (E) The percentage of CD4+ and CD8+ Tcells that have infiltrated the CD19− and CD19+ tumor after i.v. andintratumorally injection (n=3, error bars=SEM, significance determinedstudent's t-test, n.s. p>0.05).

FIG. 102. SynNotch Receptors are Versatile Regulators that Allow T Cellsto Monitor and Selectively Modulate their Microenvironment.

(A) synNotch receptors are versatile regulators of T cell response:synNotch receptors can drive diverse behaviors in primary human T cells.synNotch receptors can drive custom cytokine production profiles,effectively deliver non-native therapeutics, and control T celldifferentiation, all in an antigen-dependent and T cell activationindependent manner. (B) synNotch are sufficient to target T cells invivo to locally produce a therapeutic payload.

Example 5: Precision Tumor Recognition by Therapeutic T Cells thatIntegrate Synthetic Notch and Chimeric Antigen Receptor Signaling toDetect Combinatorial Antigen Signatures Material and Methods

The following materials and methods apply to the results described inExample 5 unless otherwise indicated.

synNotch Receptor and Response Element Construct Design

synNotch receptors were built by fusing the CD19 scFv, LaG17 (loweraffinity), or LaG16_2 (high affinity) nanobody to the mouse Notch1(NM_008714) minimal regulatory region (Ile1427 to Arg1752) and Gal4VP64or TetR VP64 (tTa). All synNotch receptors contain an n-terminal CD8asignal peptide (MALPVTALLLPLALLLHAARP (SEQ ID NO:129)) for membranetargeting and a myc-tag (EQKLISEEDL (SEQ ID NO:75)) for easydetermination of surface expression with a-myc A647 (cell-signaling#2233). The receptors were cloned into a modified pHR′ SIN:CSW vectorcontaining a PGK promoter for all primary T cell experiments. The pHR′SIN:CSW vector was also modified to make the response element plasmids.Five copies of the Gal4 DNA binding domain target sequence(GGAGCACTGTCCTCCGAACG (SEQ ID NO:130)) were cloned 5′ to a minimal CMVpromoter. Also included in this the response element plasmids is a PGKpromoter that constitutively drives mCherry expression so transduced Tcells can be easily distinguished. For all inducible CAR vectors, theCARs were tagged c-terminally with GFP and were cloned via a BamHI sitein the multiple cloning site 3′ to the Gal4 response elements. Allconstructs were cloned via In-Fusion cloning (Clontech #ST0345)).

Primary Human T Cell Isolation and Culture

Primary CD4+ and CD8+ T cells were isolated from anonymous donor bloodafter apheresis by negative selection (STEMCELL Technologies #15062 &15063). Blood was obtained from Blood Centers of the Pacific (SanFrancisco, Calif.) as approved by the University Institutional ReviewBoard. T cells were cryopreserved in RPMI-1640 (UCSF cell culture core)with 20% human AB serum (Valley Biomedical Inc., #HP1022) and 10% DMSO.After thawing, T cells were cultured in human T cell medium consistingof X-VIVO 15 (Lonza #04-418Q), 5% Human AB serum and 10 mM neutralizedN-acetyl L-Cysteine (Sigma-Aldrich #A9165) supplemented with 30 units/mLIL-2 (NCI BRB Preclinical Repository) for all experiments.

Lentiviral Transduction of Human T Cells

Pantropic VSV-G pseudotyped lentivirus was produced via transfection ofLenti-X 293T cells (Clonetech #11131D) with a pHR′ SIN:CSW transgeneexpression vector and the viral packaging plasmids pCMVdR8.91 and pMD2.Gusing Fugene HD (Promega #E2312). Primary T cells were thawed the sameday, and after 24 hours in culture, were stimulated with Dynabeads HumanT-Activator CD3/CD28 (Life Technologies #11131D) at a 1:3 cell:beadratio. At 48 hours, viral supernatant was harvested and the primary Tcells were exposed to the virus for 24 hours. At day 4 post T cellstimulation, Dynabeads were removed and the T cells expanded until day 9when they were rested and could be used in assays. T cells were sortedfor assays with a FACs ARIA II.

Cancer Cell Lines

The cancer cell lines used were K562 myelogenous leukemia cells (ATCC#CCL-243), Daudi B cell lymphoblasts (ATCC #CCL-213), and HCT115 coloncancer cells (ATCC #CCL-247). K562s were lentivirally transduced tostably express human CD19 at equivalent levels as Daudi tumors. CD19levels were determined by staining the cells with α-CD19 APC (Biolegend#302212). K562s were also transduced to stably express surface GFP (GFPfused to the PDGF transmembrane domain). All cell lines were sorted forexpression of the transgenes.

In Vitro Stimulation of synNotch T Cells

For all in vitro synNotch T cell stimulations, 2×10⁵ T cells wereco-cultured with sender cells at a 1:1 ratio. After mixing the T cellsand sender cells in round bottom 96-well tissue culture plates, thecells were centrifuged for 1 min at 400×g to force interaction of thecells and the cultures were analyzed at 24 hours for markers ofactivation (e.g. CD69) for CD4+ T cells and specific lysis of targettumor cells for CD8+ T cells with a BD LSR II. All flow cytometryanalysis was performed in FlowJo software (TreeStar).

Luminex MAGPIX Cytokine Quantification

Primary CD4+ T cells expressing the α-CD19 synNotch Gal4VP64 receptorand 5×Gal4 response elements controlling the a CAR were stimulated asdescribed above with the indicated target cancer cell line. Thesupernatant was collected at 24 hours and analyzed with a Luminex MAGPIX(Luminex Corp.) Human Cytokine Magentic 25-plex Panel (Invitrogen ref#LHC0009M) according to the manufacturer's protocol. All cytokine levelswere calculated based on standard curves with xPONENT software (LuminexCorp.).

IL-2 ELISA and CD69 Staining

CD4+ synNotch AND Gate T cells were stimulated with the indicated cancercell line as described above for 24 hours and supernatant was harvested.IL-2 levels in the supernatant were determined via IL-2 ELISA(eBiosciences #BMS2221HS). The T cells were also collected and stainedwith α-CD69 APC (Biolegend #310910) to determine if they were activated.

Assessment of synNotch AND Gate T Cell Cytotoxicity

CD8+ synNotch AND Gate T cells were stimulated for 24 hours as describedabove with target cells expressing the indicated antigens. The level ofspecific lysis of target cancer cells was determined by comparing thefraction of target cells alive in the culture compared to treatment withuntransduced T cell controls. Cell death was monitored by uptake of thelive/dead stain SYTOX Blue and shifting of the target cells out of thenormal SSC/FSC region normally populated by the target cells (ThermoScientific #S34857).

In Vitro Quantification of Luciferase Reporter Activity in synNotch TCells

Sorted CD4+ and CD8+ primary human T cells engineered to express thea-GFP nanobody (LaG17) synNotch Gal4VP64 receptor and the correspondingresponse elements controlling α-CD19 4-1BBζ CAR IRES effluc expressionwere stimulated with GFP+ or GFP− K562 cells for 24 hours (2×10⁵ T cellsand 2×10⁵ K562s). Production of effluc was assessed with the ONE-gloLuciferase Assay System (Promega #E6110). Bioluminescence was measurewith a FlexStation 3 (Molecular Devices).

In Vivo Luciferase Imaging of synNotch T Cells

Animal studies were conducted with the UCSF Preclinical TherapeuticsCore under a protocol approved by the UCSF Institutional Animal Care andUse Committee. Ten days prior to T cell injection Daudi tumors andsurface GFP Daudi tumors were injected subcutaneously into the left andright of NOD scid gamma (NSG) mice (female, 8-12 weeks old, JacksonLaboratory #005557). Sorted CD4+ and CD8+ primary human T cellsengineered to express the a-GFP nanobody (LaG17) synNotch Gal4VP64receptor and the corresponding response elements controlling α-CD194-1BBζ CAR IRES effluc expression were injected at a 1:1 CD4+ to CD8+ Tcell ration (1×10⁶ of each T cell type) i.v. into the tumor bearing mice10 days post tumor implantation. Luciferase expression was monitoredover 11 days with bioluminescent imaging performed using the IVIS 100(Xenogen) preclinical imaging system at the indicated time point. Imageswere acquired 10 min following i.p injection 150 mg/kg of D-luciferin(Gold Technology #LUCK-100). Quantification of integratedbioluminescence intensities were quantified in ImageJ (NIH).

In Vivo Dual Antigen Tumor Targeting by synNotch AND Gate T Cells

NSG mice were implanted with 5×10⁶ CD19 K562s and GFP/CD19 K562 tumorcells subcutaneously on the left and right flank, respectively. Fourdays post tumor implantation, 1×10⁶ primary human CD4+ and CD8+ T cells(2×10⁶ total T cells) engineered with the a-GFP synNotch Gal4VP64receptor and the corresponding response elements regulating α-CD194-1BBζ CAR expression or untransduced T cells were injected i.v. intothe mice. Tumor size was monitored by the UCSF Preclinical TherapeuticsCore staff via caliper over 20 days after T cell injection. ForKaplan-Meier experiments the same protocol was used, but single tumorswere injected. Mice were considered dead when the tumor size reachedeuthanasia criteria (2000 mm³).

Statistical Analysis and Curve Fitting

Statistical significance was determined by Student's t test (two-tailed)unless otherwise noted. All statistical analysis and curve fitting wasperformed with Prism 6 (Graphpad) and p values are reported(n.s.=p>0.05, *=p≤0.05, **=p≤0.01, ***=p≤0.001, ****=p≤0.0001). Allerror bars represent either S.E.M. or S.D.

Results

Design of a Two Antigen AND-Gate Circuit: synNotch Receptor Induces CARExpression

The design of a simple two receptor AND gate circuit is outlined in FIG.108D. A T cell is engineered to basally express a synNotch receptor thatrecognizes antigen A. In addition, the gene for a CAR that recognizesantigen B would also be inserted into the cell, but it would be underthe control of a promoter that requires activation by the synNotchinduced transcription factor (synNotch engagement results in receptorcleavage and release of a transcriptional activation domain, see Example3). Thus, no CAR expression or activity should be present in the celluntil the synNotch receptor is activated. This sequential receptoractivation circuit should be highly modular in design, since the antigenrecognition properties of both receptors can be easily changed byswapping extracellular domains.

Testing synNotch-Gated CAR Expression in Jurkat T Cells—CombinatorialAntigen Requirement for Jurkat T Cell Activation

To test this method of utilizing synNotch receptors to control theexpression of CARs, an attempt was made to engineer combinatorialantigen control over the activation of Jurkat T cells. In theseexperiments two model tumor antigens, CD19 and Mesothelin, weretargeted. The Jurkat T cells were engineered with an α-CD19 synNotchreceptor bearing an intracellular tetracycline-controlled transactivator(tTa) domain. α-Mesothelin 4-1BBζ CAR gene was inserted, under thecontrol of a promoter with the corresponding tetracycline responseelements (TRE) activated by the synNotch receptor (FIGS. 109A and 109B).The engineered Jurkats were co-cultured in vitro with target K562myelogenous leukemia cells with ectopic expression of CD19, Mesothelin,or both antigens together (FIG. 109A). Since these engineered Jurkatcells only express the α-Mesothelin CAR in response to α-CD19 synNotchstimulation, the T cells should not activate in response to Mesothelinalone. If the T cells are exposed to CD19, the a-Mesothelin CAR isexpressed and the T cells are primed for activation (FIGS. 109A and 109Band FIG. 114A). The T cells can then sense Mesothelin and activate.

When these cells were tested, activation only by the tumor cells thatexpressed both CD19 and mesothelin was indeed observed, as measured bythe upregulation of the activation marker CD69 and secretion of IL-2(FIGS. 109C and 109D and FIG. 114A-114B). Tumor cells expressing eithersingle antigen did not lead to activation. In the case of dual antigenstimulation, Jurkat synNotch AND Gate T cells were seen to be able toupregulate CAR expression in response to tumor cells within 6 hours andreach their peak of activation by 24 hours (FIGS. 114A and 114B). The Tcell activation (monitored via CD69) occurs shortly after CAR expressioninitiates, with an expected additional delay of a few hours (FIG. 114B).

To further characterize the dynamics of synNotch induced CAR expression,the synNotch T cells were exposed to a surrogate of the priming antigenCD19. Since the α-CD19 synNotch receptor has a myc-tag on itsextracellular domain, it was found that the receptor could also beactivated by exposure of the cells to α-myc antibody coated plates. Thisactivation approach allows for rapid cessation of synNotch activation byremoving cells from the plate-bound antigen. After 24 hours of stimuluswith α-myc antibody, the T cells were removed and the decay ofα-Mesothelin CAR expression was monitored over 24 hours. The T cellscompletely downregulated CAR expression to unstimulated levels by 24hours (half-life of expression=8 hours) (FIGS. 114C and 114D).

SynNotch-Gated CAR Expression in Human Primary T Cells—CombinatorialAntigen Control Over T Cell Activation and Tumor Killing

Given the success of the synNotch AND gate in Jurkat T cells, it wastested whether the same type of synNotch-driven CAR expression circuitcould function in primary T cells to discriminate multiple antigens. Itwas found that a spectrum of different synNotch Gal4VP64 receptorsexpress well in primary T cells and that the Gal4 response elements haveminimal basal activity in primary T cells. This is an ideal scenario forthe synNotch driven CAR expression AND gate, because there should be nobasal expression of the activating CAR until the T cells sense thesynNotch antigen.

As a proof of principal demonstration of this approach, the a-GFPnanobody synNotch Gal4VP64 receptor (recognizes surface expressed GFP)to drive expression of the α-CD19 4-1BBζ CAR (FIG. 110A) was utilized.The rationale for choosing this model setup is that the α-CD19 CAR is agold standard in the field of immunotherapy and it shows potent tumorclearance in vivo.

Human primary CD4+ T cells were engineered with the a-GFP nanobodysynNotch Gal4VP64 receptor and the corresponding response elementscontrolling α-CD19 4-1BBζ CAR expression, then exposed to K562 targetcells expressing CD19 only, GFP only, or GFP and CD19. The CD4+ T cellsonly displayed expression of the α-CD19 4-1BBζ CAR when stimulated withcells expressing the synNotch ligand, GFP (FIG. 110B). Moreover, these Tcells only showed activation, as assayed by cytokine production, whenexposed to target cells expressing both antigens, GFP and CD19, on theirsurface (FIGS. 110B and 110C).

Human primary CD8+ cells containing the same dual receptor circuit alsoshowed AND gate behavior, only killing targets when GFP and CD19 werepresent on the target cell surface (FIG. 110D-110F). Thus, the synNotchAND gate is functional in the critical cell types required for T cellimmunotherapy in humans. To show the versatility and modularity of thisapproach, three other synNotch/CAR AND gate configurations were tested.All showed combinatorial antigen requirements for CD4+ and CD8+ T cellactivation (FIG. 115A-115I).

synNotch Receptors Drive Tumor Localized CAR Expression In Vivo

Since synNotch receptors reliably gate CAR expression in primary T cellsin vitro, it was tested whether T cells could be targeted to tumors invivo via synNotch receptors and only express the CAR when in the tumormicroenvironment. For this experiment, bilateral xenograft CD19 Daudi Bcell lymphoblast tumors were injected into immunocompromised NOD scidIL-2Rγ^(−/−) (NSG) mice. Wild-type Daudi cells (containing no synNotchligand) were injected subcutaneously in the left flank, while Dauditumor cells expressing surface GFP were injected in the right flank.After giving the tumors ten days to implant, primary CD4+ and CD8+ humanT cells equipped with the a-GFP synNotch Gal4VP64 receptor and thecorresponding response elements controlling the expression of the α-CD194-1BBζ CAR and an IRES enhanced firefly luciferase (effluc) reporter(FIGS. 116A and 116B) were injected. Luciferase expression was monitoredas a reporter for CAR expression over the course of 11 days (FIG. 111A,FIG. 116C). The T cells started to express the CAR selectively in theGFP+ Daudi tumor by day 1 and continually increased local expression ofthe CAR over the 11 day period in the dual antigen tumor (FIGS. 111B and111C). The increase in luciferase signal in the target tumor is likely acombination of synNotch-driven CAR expression and expansion of cells inthe dual antigen target tumor (FIG. 116B). No increase in luciferase wasobserved in the control GFP− tumor.

Highly Selective Combinatorial Antigen Tumor Clearance In Vivo bysynNotch Gated CAR Expression

It was shown that the a-GFP synNotch receptor could target T cells totumors and control local expression of the α-CD19 CAR. It was thentested whether the synNotch AND gate T cells could selectively clear adual antigen tumor in vivo. For these experiments, a similar bilateraltumor model was set up with K562 tumor cells (FIG. 112A and FIGS. 117Aand 117B). The tumors were implanted and 4 days were allowed forimplantation (K562 tumors grow more rapidly and establish large tumorscompared to Daudi cells). At day 4, CD4+ and CD8+ T cells bearing thea-GFP synNotch→α-CD19 CAR AND gate circuit were injected, and tumorgrowth was monitored via caliper for 20 days (FIG. 112A). A group ofmice was treated with untransduced control T cells to have a referencefor tumor growth. In this experiment, the T cells are directlychallenged to discriminate dual antigen “disease” tumors from singleantigen “bystander” tissues, all within the same animal.

The synNotch AND gate T cells displayed remarkably high and reproduciblediscriminatory action against the two tumors in present in the sameanimal. In all animals they selectively cleared the dual antigen“disease” tumor (GFP/CD19+) while leaving the single antigen “bystander”tumor (CD19+ only) unperturbed. These bystander single antigen tumorsgrew at rates similar to the negative control tumor treated withuntransduced T cells (FIG. 112B, 112C, 117B). Thus there is littledetectable OFF-target killing of the “bystander” single antigen tumor.

Single tumor experiments were set up, where mice were implanted witheither a single antigen (CD19+ only) or a dual antigen (GFP/CD19+) K562tumor. The mice were then treated with synNotch AND gate T cells oruntransduced control T cells. The mice treated with control T cells allreached euthanasia criteria rapidly regardless of the tumor type. Themice with GFP/CD19 tumors treated with synNotch AND gate T cells alllived and the tumor was completely cleared by day 25 post tumorinjection (FIG. 112D). Mice with CD19 only tumors treated with synNotchAND gate T cells reached euthanasia criteria at the same rate as micetreated with untransduced T cells suggesting there is no OFF-targetkilling of the single antigen tumor (FIG. 112D). These in vivo datacollectively show that synNotch-gated CAR expression is an effective ANDgate allowing T cells to confine their activity to the tumormicroenvironment and only activate and kill in response to multipleantigens.

One concern was whether the AND-gate T cells could engage a tumorexpressing the synNotch ligand (GFP), become primed by expressing theα-CD19 CAR, then migrate elsewhere to then kill single antigen (CD19+only) bystander tissues. To test this, experiments were performed with abilateral tumor model, but in this case used one tumor with CD19+ onlycells and the other tumor with GFP+ only cells (i.e. two single antigentumors) (FIG. 117D). In these mice, it would be possible for the T cellsto be primed by the GFP+ only tumor, then kill the CD19+ only tumors.When growth of the CD19+ tumor was monitored using the AND gate cells,it was found that it was identical to the growth observed when treatedwith negative control T cells (untransduced). Thus, there appears to beno evidence for priming of the AND gate T cells and subsequent killingof bystander CD19+ cells elsewhere. It was hypothesized that in theseAND gate T cells, the decay rate of induced CAR expression, is likelyfaster or comparable to the rate of migration out of the priming tumor,which would explain the requirement for highly local dual antigens.

FIG. 108. synNotch Receptors for Combinatorial Antigen Sensing in TCells

(A) CAR or tumor-specific TCR T cells generally target single antigens,thereby often causing OFF-target tissue damage. Improved therapeutic Tcells will require multiple sensors that recognize combinations of bothtumor antigens and tissue-specific antigens, allowing the cells toassess their environment and make more precise decisions on when toactivate. Such therapeutic cells would be better equipped to distinguishtarget diseased tissue from normal tissue. (B) New types of receptorsthat sense combinations of antigens and regulate T cell signaling andtranscription must be built to allow for sophisticated cellulardecision-making and more precise therapeutic T cell responses. (C)synNotch receptors are engineered with a custom extracellularligand-binding domain such as an scFv or nanobody directed towards anantigen of interest (e.g. tumor or tissue specific antigen). Upon ligandrecognition by the synNotch receptor, an orthogonal transcription factor(e.g. TetRVP64 or Gal4VP64) is cleaved from the cytoplasmic tail thatregulates a custom genetic circuit. (D) Design of a synNotch AND-gatecircuits, which requires T cells to sense two antigens to activate. ThisAND-gate signaling circuit works in two sequential steps: 1) A synNotchreceptor allows the T cell to recognize the first antigen A and 2) the Tcell expresses a CAR directed towards a second tumor antigen B. If A andB are present, the T cells can activate and kill the target tumor.

FIG. 109. synNotch-Gated CAR Expression—Combinatorial AntigenRequirement for Jurkat T Cell Activation

(A) Engineering a two receptor AND-gate circuit: α-CD19 synNotchreceptor induces a-mesothelin CAR expression. (B) Jurkat T cells wereengineered with the α-CD19 synNotch tTa receptor and the correspondingresponse elements controlling a-mesothelin CAR expression. The Jurkat Tcells must first recognize CD19 on the target tumor via their synNotchreceptor to initiate CAR expression. After the T cell is primed toactivate by CD19, the a-mesothelin CAR can then bind mesothelin andactivate the Jurkat cell. Two canonical markers of T cell activation areCD69 upregulation and IL-2 production. The synNotch AND gate Jurkat Tcells should only activate when exposed to target tumor cells expressingboth CD19 and Mesothelin. (C) Histograms of the activation marker CD69in synNotch AND Gate Jurkat T cells co-cultured with single antigen(Mesothelin only) or dual antigen (CD19/Mesothelin) K562 tumor cells for48 hours. CD69 was only expressed when the T cells were exposed to dualpositive K562s (representative of 3 independent experiments)(D) IL-2ELISA showing IL-2 production by synNotch AND-Gate Jurkats only whenexposed to dual antigen K562s (n=3, error bars are SEM, significancedetermined by Student's t-test, ****=p≤0.0001)

FIG. 110. synNotch Gated CAR Expression in Human Primary TCells—Combinatorial Antigen Control Over Therapeutic T Cell Activationand Tumor Killing

(A) Human primary CD4+ and CD8+ T cells were engineered with the a-GFPnanobody synNotch Gal4VP64 receptor and the corresponding responseelements controlling expression of the α-CD19 4-1BBζ CAR. These CD4+ orCD8+ synNotch AND gate T cells first must sense surface GFP via theirsynNotch receptor and only then do they express the α-CD19 CAR and areprimed to activate. The synNotch AND gate primary T cells should onlyactivate and produce cytokine or kill target cells if they sense bothGFP and CD19. (B) Primary CD4+ synNotch AND gate T cells described inpanel A were co-cultured with CD19 only or surface GFP/CD19 K562s.Histograms of α-CD19 CAR GFP receptor expression level show that the CARis only expressed when GFP is present on the surface of the target cell(representative of at least 3 independent experiments). (C) Thesupernatant from CD4+ synNotch AND gate T cells activated either by CD19only or GFP/CD19 K562s was analyzed for the presence of a 25 cytokinesvia Luminex. Cytokines were only produced when the T cells were exposedto GFP/CD19 T cells (error bars are SEM, n=3). (D) CD8+ synNotch ANDgate primary T cells were engineered as described in panel A. As withthe CD4+ T cells, the histograms of α-CD19 CAR GFP receptor expressionlevel show that the CAR is only expressed when GFP is present on thesurface of the target cell (representative of at least 3 independentexperiments). (E) Forward and side scatter flow cytometry plots after 24hour co-culture of CD8+ synNotch AND gate primary T cells with eitherCD19 only or GFP/CD19 tumors cells. The T cells fall within the bluegate and the target K562s are in the red gate. The synNotch AND gate Tcells only killed the GFP/CD19 K562s shown by the reduction cells in theK562 gate (representative of 3 experiments). (F) Quantificationreplicate CD8+ synNotch AND gate primary T cell cytotoxicity data shownin panel E. (n=3, error bars are SEM, significance determined byStudent's t-test *=p≤0.05).

FIG. 111. synNotch Receptors Drive Tumor Localized CAR Expression InVivo.

(A) Primary human CD4+ and CD8+ T cells were engineered with the a-GFPsynNotch Gal4VP64 receptor and the corresponding response elementsregulating α-CD19 4-1BBζ CAR IRES effluc expression and injected i.v.into mice with a Daudi tumor (CD19 only) on the left flank and a surfaceGFP Daudi (GFP/CD19) tumor on the right flank. Luciferase expression wasmonitored over 11 days after i.v. injection of engineered T cells. (B) Arepresentative image of luciferase expression in mice treated asdescribed in panel A at day 7 post T cell injection. Luciferaseexpression was high in the GFP/CD19 tumor only indicating localized CARexpression in the dual antigen tumor (n=2 mice)(C) Quantification ofintegrated intensity of luciferase levels in the left flank Daudi tumor(CD19 only) and surface GFP Daudi tumor (GFP/CD19) in the right flank.Luciferase expression is enriched in the dual antigen tumor at all timepoints (error is SD n=2).

FIG. 112. Selective Combinatorial Antigen Tumor Killing In Vivo bysynNotch Gated CAR Expression

(A) Primary human CD4+ and CD8+ T cells were engineered with the a-GFPsynNotch Gal4VP64 receptor and the corresponding response elementsregulating α-CD19 4-1BBζ CAR expression and were injected i.v. into micewith a CD19 K562s on the left flank and a surface GFP/CD19 K562 tumor onthe right flank. Tumor size was monitored over 16 days after i.v.injection of engineered T cells or untransduced T cell controls. (B)Graphs showing CD19 and GFP/CD19 tumor volumes for mice treated withsynNotch AND gate T cells (top) and untransduced control T cells(bottom). synNotch AND gate T cells target the dual antigen tumorexclusively and the CD19 only tumor grew at the same rate as in micetreated with untransduced control T cells (n=5 mice, error bars are SEM,significance determine by Student's t-test **=p≤0.01, ***=p≤0.001)(C)Tumor volume measurement for individual mice treated with synNotch ANDgate T cells. All mice showed selective killing of the dual antigentumor. (D) Kaplan-Meier graphs showing synNotch AND gate T cells clearGFP/CD19 tumors with 100% of the mice surviving. Mice with CD19 onlytumors are not cleared by synNotch AND gate T cells and haveuncontrolled tumor growth. The corresponding tumor growth curves aregiven on the right of panel D (n=5 mice, error bars are SEM,significance determine by Student's t-test **=p≤0.01).

FIG. 113. synNotch Receptors Control and Localize CAR T Cell Responsefor Precision Immunotherapy.

(A) T cells were engineered with (genetically modified to produced)synNotch receptors that sense tumor antigens and upregulate expressionof a CAR to a second antigen. Thus, these synNotch AND gate T cells onlyactivate in response to combinatorial antigen recognition in the tumormicroenvironment, preventing OFF-target toxicity mediated by singleantigen recognition. (B) synNotch AND gate T cells unlike therapeutic Tcells that target single antigens can reliably discriminatecombinatorial antigen targets from single antigen bystander tissue.Combinatorial antigen sensing by synNotch-CAR T cells could aid inprecisely targeting T cells to tumors preventing OFF-target toxicity.(C) Expansion of targetable antigen space. Tumor-specific antigens arerare compared to tumor-associated antigens (antigens that are expressedon normal tissue but are more highly expressed on tumors). Since CARsfully activate T cells resulting in the killing of target tissue, singleCAR T cells must be targeted to tumor-specific antigens in order toreduce fatal OFF-target toxicity (top Venn diagram). synNotch receptorscan gate CAR expression and control where the T cells are armed. Whentargeting tumor specific antigen combinations, it may now be possible touse CAR receptors directed towards tumor-associated antigens. Thisshould reduce OFF-target damage to tissues that express the CAR antigenin other parts of the body.

FIG. 114. synNotch-Gated CAR Expression—Combinatorial AntigenRequirement for Jurkat T Cell Activation.

(A) α-CD19 synNotch Jurkat T cells controlling expression of thea-mesothelin CAR fused to GFP were incubated for 48 hours with Meso onlyor CD19 and Meso+K562s. Histograms of a-mesothelin CAR GFP levels showthat the CAR is only expressed when the Jurkats are exposed to CD19. (B)Plots of normalized a-mesothelin CAR GFP and CD69 levels calculated fromhistograms in panel A and FIG. 2B. The half time for maximal expressionof the CAR and CD69 was 6 hours and 13 hours respectively. (C) synNotchAND gate Jurkat T cells were stimulated with plate-bound α-myc antibodythat binds a myc-tag on the extracellular domain of the synNotchreceptor for 24 hours. After 24 hours the cells were removed from thea-myc stimulus and CAR expression was monitored for 24 hours. (D)Histograms of a-mesothelin CAR GFP expression after removal of thesynNotch stimulus. Normalized CAR is plotted and fit to a one-phasedecay showing a 8 hour half-time of down regulation.

FIG. 115. synNotch Gated CAR Expression in Human Primary TCells—Combinatorial Antigen Control Over Therapeutic T Cell Activationand Tumor Killing

(A) CD4+ primary T cells were engineered with the α-CD19 synNotchGal4VP64 receptor and the corresponding response elements controllingα-Mesothelin 4-1BBζ CAR EGFP expression. The T cells were thenco-cultured with Mesothelin only, CD19 only, or CD19/Mesothelin K562sfor 24 hours and CD69 upregulation and IL-2 production were assayed. (B)Histograms showing α-Mesothelin CAR EGFP levels and CD69 levels on CD4+synNotch primary T cells cultured as described in panel A. Theα-Mesothelin CAR was only expressed when CD19 was on the target K562sand the T cells only expressed the activation marker CD69 when both CD19and Mesothelin were on the target K562s (representative of 3experiments). (C) IL-2 levels from supernatant harvested from culturesdescribed in panel A. IL-2 was only produced when the T cells wereexposed to targets cell expressing both CD19 and Mesothelin (n=3, errorbars are SEM, significance determined by Student's t-test ***=p≤0.001).(D) CD8+ primary human T cells were engineered as described in panel A.For CD8+ T cells specific cytotoxicity of Mesothelin only, CD19 only, orCD19/Mesothelin target K562s was determined. The synNotch AND gate CD8+T cells should only kill dual positive K562s. (E) Histograms showingα-Mesothelin CAR EGFP levels on CD8+ synNotch primary T cells culturedas described in panel A. The α-Mesothelin CAR was only expressed whenCD19 was on the target K562s (representative of 3 experiments). (F)Quantification replicate CD8+ synNotch AND gate primary T cellcytotoxicity showing specific killing of target K562s with both CD19 andMesothelin expression (n=3, error bars are SEM, ***=p≤0.001). (G) CD4+primary T cells were engineered with the a-GFP nanobody synNotchGal4VP64 receptor and the corresponding response elements controllingα-Mesothelin 4-1BBζ CAR EGFP expression. The T cells were thenco-cultured with Mesothelin only, GFP only, or GFP/Mesothelin K562s for24 hours and CD69 upregulation and IL-2 production were assayed. (H)Histograms showing a-Mesothelin CAR EGFP levels and CD69 levels on CD4+synNotch primary T cells cultured as described in panel G. Theα-Mesothelin CAR was only expressed when GFP was on the target K562s andthe T cells only expressed the activation marker CD69 when both GFP andMesothelin were on the target K562s (representative of 3 experiments).(I) IL-2 levels from supernatant harvested from cultures described inpanel G. IL-2 was only produced when the T cells were exposed to targetscell expressing both GFP and Mesothelin (n=3, error bars are SEM,****=p≤0.0001).

FIG. 116. synNotch Receptors Drive Tumor Localized CAR Expression InVivo.

(A) Representative dot plots showing expression of the a-GFP synNotchGal4VP64 receptor and the corresponding response elements regulatingα-CD19 4-1BBζ CAR IRES effluc in primary CD4+ and CD8+ T cells. The Tcells outlined by the red box were sorted and used for in vivo and invitro experiments. (B) Bar graph showing luciferase activity in synNotchAND CD4+ and CD8+ T cells from panel A after exposure for 24 hours withGFP− or GFP+K562s. Luciferase was specifically expressed in response toGFP (n=3, error bars are SEM, ****=p≤0.0001). (C) Tumor growth curvesare given for mice analyzed in FIG. 4C.

FIG. 117. Selective Combinatorial Antigen Tumor Killing In Vivo bysynNotch Gated CAR Expression

(A) Representative dot plots engineered showing the expression of theα-GFP synNotch Gal4VP64 receptor and the corresponding response elementsregulating α-CD19 4-1BBζ CAR in primary human CD4+ and CD8+ T cells. TheT cells red boxed quadrant were sorted and used for experiments in FIG.112. (B) Flow cytometry plots showing the expression of CD19 (purple)and GFP and CD19 (green) in K562s utilized for in vitro and in vivoexperiments. (C) Tumor growth curves for individual mice with bilateralCD19 (left flank) and GFP and CD19 (right flank) tumors treated withcontrol untransduced CD4+ and CD8+ T cells. The data underlies FIG. 5Blower panel. (D) Primary human CD4+ and CD8+ T cells were engineeredwith the α-GFP synNotch Gal4VP64 receptor and the corresponding responseelements regulating α-CD19 4-1BBζ CAR expression and were injected i.v.into mice with a CD19 K562s on the left flank and a surface GFP K562tumor on the right flank to test if the T cells migrate from theGFP+‘priming tumor’ and kill the OFF-target CD19 only tumor. Tumor sizewas monitored over 16 days after i.v. injection of engineered T cells oruntransduced T cell controls. (E) Graph showing CD19 tumor volumes formice treated with synNotch AND gate T cells (solid line) or untransducedcontrol T cells (dotted line). The CD19 tumor is not targeted suggestingthere is no migration of primed T cells from the GFP+ tumor (n=5, errorbars are SEM, no significant difference at any timepoints based onstudent's t test, p>0.05).

Example 6: SynNotch Induced Expression of Foxp3 in Human CD4 T Cells

Direct intracellular staining of Foxp3 was used to measure Foxp3induction in human CD4 T cells expressing anti-CD19 synNotch. Anti-CD19synNotch expressing CD4 T cells were stimulated with either CD19+(CD19positive) K562 cells or CD19− (CD19 negative) K562 cells. Foxp3 wasinduced in CD19 synNotch cells when stimulated with the CD19+K562 cells(FIG. 118, “cN Foxp3+stim K562”). In comparison, induction was not seenwhen the CD19 synNotch cells were stimulated with CD19− K562 cells (FIG.118, “cN Foxp3+irrelevant K562”). Untransduced cells were used as anegative control (FIG. 118, “untransduced”).

Induction of Foxp3, a master regulator of the regulatory pathway in thedevelopment and function of regulatory T cells, using a CD19 responsiveSynNotch demonstrates local antigen driven induction of regulatory Tcell fate.

Example 7: Controlled Antibody Production by Anti-CD19 synNotchExpressing CD4 T Cells

Antibody expression constructs were designed for human T cell synNotchinduced antibody secretion. A schematic showing the general antibodyconstruct design is provided in FIG. 119. In FIG. 119, from left toright, the domains of the general construct design are human IgG heavychain signal peptide, Myc tag, VH domain, CH domain, furin cleavagesite, SGSG spacer, T2A sequence, human IgG light chain signal peptide,HA tag, VL domain and CL domain. Constructs were designed for synNotchinduced secretion of the following antibodies: pembrolizumab(anti-PD-1), Tremelimumab (anti-CTLA4) and 9E10 (anti-myc).

A general scheme for an in vitro assay used to test SynNotch inducedantibody secretion is provided in FIG. 120. Briefly, CD4 T cells weretransduced with an anti-CD19 synNotch construct that, when activated,drives expression from an introduced antibody construct (see e.g., FIG.119) and subsequent secretion of the antibody. Expression of theantibody construct is activated by the anti-CD19 synNotch intracellulardomain, in this case a tTA intracellular domain. In the assay, suchtransduced T cells were contacted with either CD19 expressing (CD19+;also referred to as “synNotch ligand+”) K562 target cells oruntransduced (CD19-) K562 target cells. The transduced CD4 T cells andthe K562 target cells were co-cultured for 24, 48 and 72 hours prior tocollection of culture supernatant.

Antibody secreted by the anti-CD19 synNotch CD T cells was quantified bya cell surface “Sandwich ELISA” flow cytometry assay (see FIG. 121).Briefly, cells expressing the antibody ligand (“antibody ligand+ targetcell”), i.e., cells expressing PD-1 in the case of the pembrolizumabantibody assay, were subjected to a 10 min. human Fc Block. Followingblocking, 50 1 of serially diluted supernatant from either CD19+ orCD19− target cell co-culture was added and incubated with the cells for30 min. Following primary antibody incubation, the cells were washedthree times and 50 1 of fluorescent conjugated secondary antibody wasadded. In the instant case, anti-myc-AF647 was used as the secondaryantibody and the secondary incubation was performed for 30 min.Following incubation with secondary antibody, the cells were washedthree times and then subjected to analysis with a BD LSR II flowcytometer.

The results of this analysis for SynNotch T cells modified to secretepembrolizumab are provided in FIG. 122. From top to bottom in FIG. 122,the results correspond to the following test groups:

TABLE 1 Group Stimulus Primary Incubation Co-culture Time 1 CD19+ K562cells Ligand+ K562 72 hours supernatant 2 CD19− K562 cells UntransducedK562 72 hours supernatant 3 CD19+ K562 cells Ligand+ K562 48 hourssupernatant 4 CD19− K562 cells Untransduced K562 48 hours supernatant 5CD19+ K562 cells Ligand+ K562 24 hours supernatant 6 CD19− K562 cellsUntransduced K562 24 hours supernatant 7 Not Applicable PBS NotApplicable

PD-1 expressing target cells showed high anti-myc-AF674 secondarystaining when incubated with supernatant from anti-CD19 synNotch T cellsco-cultured with CD19 expressing K562 cells (“Ligand+K562 supernatant”)which increased with increasing co-culture time (24h, 48h and 72 hr). Incomparison, PD-1 expressing target cells showed low anti-myc-AF674secondary staining when incubated with supernatant from anti-CD19synNotch T cells co-cultured with untransduced (CD19-) K562 cells(“untransduced K562 supernatant”).

This data demonstrates that anti-CD19 synNotch T cells were induced byCD19 expressing (CD19+) K562 cells to express and secrete theheterologous pembrolizumab antibody. Correspondingly, anti-CD19 synNotchT cells were not induced to secrete pembrolizumab when co-cultured withuntransduced K562 cells. FIG. 139 provides the results of a similaranalysis of SynNotch CD4 T cells modified to secrete pembrolizumab withan alternative expression construct, UAS_Prembro_IRES_mC_pGK_tBFP. FIG.140 provides the results of a similar analysis of SynNotch E6-1 Jurkatcells modified to secrete pembrolizumab using the construct of FIG. 138.FIG. 141 provides the results of a similar analysis of SynNotch E6-1Jurkat cells modified to secrete pembrolizumab using the alternativeconstruct of FIG. 139. FIG. 142 and FIG. 143 provides the results of asimilar analysis of SynNotch CD4 T cells and E6-1 Jurkat cells,respectively, modified to secrete Tremelimumab (anti-CLTA4) withUAS_tremelimumab_pGK_mC expression construct. Each row of data presentedin FIGS. 139-143 generally corresponds to the groups presented above inTable 1.

Accordingly, SynNotch can be used to induce production of heterologousantibodies in human CD4 T cells where the antibody is produced only whenthe SynNotch cell is stimulated by the corresponding antigen (e.g.,CD19). As such, in one embodiment, the SynNotch system can be used todirect the local production of therapeutic antibodies in response torecognition of a specific target antigen.

Example 8: Split SynNotch Modulation with an Adapter Molecule

A split SynNotch signaling system was constructed having two differentanti-GFP nanobodies that bind different epitopes of GFP where onenanobody (“LaG2”) is expressed on an “anchor cell”, the other nanobody(“LaG17”) is expressed on a “receiver cell” as a portion of a SynNotchreceptor and GFP serves as a soluble adapter (see FIG. 123). The LaG17nanobody SynNotch receptor was engineered to have a tTa intracellulardomain that, upon activation, induces expression of an mCherry reporterfrom a TRE expression construct. Accordingly, in the designed splitSynNotch system addition of the soluble adapter molecule (i.e., GFP)induces SynNotch signaling in the receiver cell only in the presence ofthe anchor cell.

The components of the described split SynNotch system were expressed intheir corresponding cells in co-culture. Specifically, surface boundLaG2 anti-GFP nanobody was expressed in L929 cells (i.e., “anchorcells”) and SynNotch with extracellular LaG17 anti-GFP nanobody andintracellular tTa was expressed in “receiver cells” having a TRE/mCherryexpression reporter cassette. As a negative control, receiver cells wereco-incubated with L929 cells that do not express the surface bound LaG2(i.e., the “anchor”). GFP was added to the co-culture medium at serialconcentrations (0 nM, 0.1 nM, 1 nM, 10 nM and 1000 nM) and mCherryexpression, as an indication of receiver cell activation, was quantifiedby flow cytometry.

Receiver cell activation was only observed in the presence of LaG2expressing anchor cells and mCherry expression was GFP “adapter” dosedependent (see FIG. 124). At 0 nM purified GFP added to receiver cellsco-incubated with L929 anchor cells expressing LaG2 the receiver cellsremained “OFF” (i.e., mCherry expression was essentially the same asthat of receiver cells co-incubated with L929 negative control cells andthe two peaks overlap). At increasing concentrations of purified solubleGFP, activation (as indicated by mCherry expression) was observed inreceiver cells co-incubated with L929 anchor cells expressing LaG2 (theright most peaks in 0.1 nM, 1 nM, 10 nM and 1000 mM GFP). Even in thepresence of increasing concentrations of GFP, receiver cellsco-incubated with L929 negative control cells were not activated andremained “OFF” (left most peaks in 0.1 nM, 1 nM, 10 nM and 1000 mM GFP).

Accordingly, receiver cell activation was dependent on the presence ofboth the partner anchor cell and a sufficient concentration of thesoluble GFP adapter molecule. Thus, receiver cell activation can becontrolled both spatially, through the local presence of the anchorcell, and conditionally by modulating the presence of a sufficientconcentration of the adapter molecule.

The split SynNotch signaling paradigm was used to further demonstrate a3 cell system where receiver cell activation is modulated by a solubleadapter produced by a cell of the system.

A schematic representation of a three cell split SynNotch signalingsystem is provided in FIG. 125. Briefly, the two cell split SynNotchsystem described above in FIG. 123 was utilized, however, a third cellwas added to the system which constitutively secretes GFP. Termed the“sender cell”, soluble GFP is expressed from an expression cassetteunder control of the constitutively active Ela promoter. Using a ratioof sender cells to receiver cells to anchor cells of 0.5:0.25:0.25,co-incubation of receiver cells with anchor cells expressing surfaceLaG2 anti-GFP nanobody in the presence of sender cells secreting GFP,activation of the receiver cells (as indicated by increased mCherryexpression) was seen (see FIG. 126, middle panel, “ON”). In comparison,when the same ratio of cells was used but the anchor cells were replacedwith negative control L929 cells that do no expression LaG2, no receivercell activation was seen (see FIG. 126, top panel, “OFF”). In addition,when the ratio was altered to contain few GFP expressing sender cells(ratio of sender cells to receiver cells to anchor cells of0.01:0.495:0.495), receiver cells were also not activated (see FIG. 126,bottom panel, “OFF”).

Accordingly, receiver cell activation was dependent on the presence ofboth the partner anchor cell and a GFP expressing send cells. Thus,receiver cell activation can be controlled both spatially, through thelocal presence of the anchor cell, and conditionally by modulating thepresence of adapter molecule expressing cells. An additional level ofcontrol is available by modulating the expression of the adaptermolecule within sender cells, e.g., by use of a regulatable promoterduring expression of the adapter molecule in the sender cell.

The three cell split SynNotch signaling system allows for multi-inputcontrol of synNotch signaling. In addition, by controlling theexpression and secretion of the diffusible adapter from the sender cell,SynNotch activation is not necessarily dependent on adjacent cell-cellcontact and may be tuned spatially based on the radius of diffusiblesecreted adapter.

The split SynNotch signaling paradigm was used to further demonstrate a3 cell system where inhibition of receiver cell activation is modulatedby a soluble adapter produced by a cell of the system.

A schematic representation of a three cell split SynNotch inhibitorysignaling system is provided in FIG. 127. The receiver cell of the twosplit SynNotch system described above in FIG. 123 was utilized. Asurface expressed GFP cell was utilized as the “sender cell” whichactivates the “receiver cell” upon binding of the surface expressed GPFto the LaG17 anti-GFP nanobody portion of the receiver cell expressedSynNotch. Similar to above, activation of the receiver cell can bequantified by measuring expression from a TRE/mCherry reporter cassette.The third cell of this system, termed the “inhibitor cell”,constitutively expresses soluble LaG16-LaG2 anti-GFP nanobody which actsas a competitive inhibitor for the binding of the surface expressed GFPof the sender cell and the LaG17 of the receiver cell. Thus,introduction of inhibitor cells to a co-culture of sender cells andreceiver cells competitively inhibits binding between the sender cellsand receiver cells, inhibiting activation of the receiver cells whichcan be measured by reduced mCherry expression.

A control culture of receiver cells only, i.e., a culture of receivercells that did not contain sender or inhibitor cells, did not result inactivation of the receiver cells (see FIG. 128, top panel “OFF”).Activation of receiver cells was seen in a co-culture containing sendercells, receiver cells and negative control inhibitor cells of theparental L929 cell line that do not express the competitive inhibitor(ratio of sender cells to receiver cells to negative control inhibitorcells of 0.25:0.25:0.5) (see FIG. 128, middle panel “ON”). When the samecell ratio was utilized (0.25:0.25:0.5) in a co-culture of sender cells,receiver cells and inhibitor cells that express the competitiveinhibitor (sLaG16-LaG2-expressing L929 cells) inhibition of receivercell activation was observed as indicated by a decrease in mCherryexpression (see FIG. 128, bottom panel “OFF”).

Accordingly, receiver cell activation, which was dependent on binding ofthe SynNotch of the receiver cell to the surface GFP of the sender cell,was inhibited by the expression of a soluble competitive inhibitorexpressed from the inhibitor cell. Thus, 3-cell regulatory systems, withboth positive and negative signaling, can be used to both controlSynNotch expressing receiver cell activation. Therefore, spatial andconditional receiver cell modulation is not limited to positive signalsand can be controlled using negative signals and combinations thereof.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1-143. (canceled)
 144. A nucleic acid encoding a chimeric polypeptidecomprising: (a) an extracellular domain that comprises an antigenbinding region of an antibody; (b) transmembrane region and one or moreprotease cleavage sites; and (c) an intracellular domain that comprisesa DNA binding domain: i. wherein the intracellular domain does notcomprise an immunoreceptor activation domain or a co-stimulatory domain;and ii. when the chimeric polypeptide is expressed in a mammalian cell,binding of the antigen binding region of the antibody to a correspondingantigen induces proteolytic cleavage of the chimeric polypeptide at oneor more of the protease cleavage sites to release the intracellulardomain.
 145. The nucleic acid of claim 144, wherein the antigen bindingregion is a scFv or a nanobody.
 146. The nucleic acid of claim 144,wherein the antigen is a cancer-associated antigen.
 147. The nucleicacid of claim 144, wherein the chimeric polypeptide comprises a Notchregulatory region between the antigen binding region of (a) and the DNAbinding domain of (c).
 148. The nucleic acid of claim 147, wherein theNotch regulatory region comprises a Lin 12-Notch repeat, aheterodimerization domain comprising an S2 proteolytic cleavage site anda transmembrane domain comprising an S3 proteolytic cleavage site. 149.The nucleic acid of claim 147, wherein the Notch regulatory regioncomprises, at its N-terminus, one or more epidermal growth factor (EGF)repeats.
 150. The nucleic acid of claim 149, wherein the Notchregulatory region comprises, at its N-terminus, 2 to 11 EGF repeats.151. The nucleic acid of claim 148, wherein the S2 proteolytic cleavagesite is an ADAM-17-type protease cleavage site comprising an Ala-Valdipeptide sequence.
 152. The nucleic acid of claim 148, wherein the S3proteolytic cleavage site is a gamma-secretase (γ-secretase) cleavagesite comprising a Gly-Val dipeptide sequence.
 153. The nucleic acid ofclaim 144, wherein the chimeric polypeptide further comprises an S1proteolytic cleavage site.
 154. The nucleic acid of claim 144, whereinthe chimeric polypeptide lacks an S1 proteolytic cleavage site.
 155. Thenucleic acid of claim 153, wherein the S1 proteolytic cleavage site is afurin-like protease cleavage site comprising the amino acid sequenceArg-X-(Arg/Lys)-Arg, where X is any amino acid.
 156. The nucleic acid ofclaim 147, wherein the Notch regulatory region has at least 85% aminoacid sequence identity to any one of the Notch regulatory regions of SEQID NO:131, 132 and 135-137.
 157. The nucleic acid of claim 144, whereinthe intracellular domain comprises a transcription factor or a firsthalf of a split transcription factor.
 158. A genetically modified cellcomprising the nucleic acid of claim 144, wherein the nucleic acid isoperably linked to a promoter and the chimeric polypeptide is expressedin the cell.
 159. The genetically modified cell of claim 158, whereinthe cell further comprises a second nucleic acid comprising atranscriptional control element that is operably linked to a codingsequence for a therapeutic agent, wherein binding of the antigen bindingregion of the chimeric polypeptide to an antigen releases theintracellular domain and activates expression of the therapeutic agent.160. The genetically modified cell of claim 159, wherein the therapeuticagent is immunosuppressive.
 161. The genetically modified cell of claim159, wherein the therapeutic agent stimulates immune cells.
 162. Thegenetically modified cell of claim 159, wherein the therapeutic agent isselected from the group consisting of: an antibody, a chemokine, achemokine receptor, a cytokine, a cytokine receptor, a chimeric antigenreceptor (CAR), a T cell receptor (TCR), an activating immunoreceptor,an inhibiting immunoreceptor, an immunoactivator, an immunoinhibitor, anapoptosis inducer, an apoptosis inhibitor, a toxin derived protein and asite-specific nuclease.
 163. The genetically modified cell of claim 159,wherein the therapeutic agent is a chimeric antigen receptor (CAR). 164.The genetically modified cell of claim 158, wherein the cell is animmune cell, a neuron, an epithelial cell, an endothelial cell, or astem cell.
 165. The genetically modified cell of claim 158, wherein thecell is a T cell, a B cell, a monocyte, a natural killer cell, adendritic cell, a macrophage, a regulatory T cell, a helper T cell, or acytotoxic T cell.
 166. The genetically modified cell of claim 158,wherein the cell is a T cell.